JP2016538333A - Human papillomavirus therapeutic vaccine - Google Patents

Abstract

Peptides and compositions for therapeutic vaccines for treating people infected with human papillomavirus are presented. Methods of using the compositions and methods of treating people who are at risk for cancer or who are infected with human papillomavirus, including those already with cancer due to human papillomavirus are presented. [Selection] Figure 1A

Description

  Cervical cancer is the fourth most common cancer in women worldwide, with 528,000 cases of disease and 266,000 deaths annually. In the United States, there are 12,360 new cases of cervical cancer each year and 4,020 deaths. The most common type of high-risk human papillomavirus is HPV16, which is a major cause of cervical cancer. Of the over 100 different types of human papillomavirus, at least 15 types are strongly associated with invasive squamous cell carcinoma of the cervix. HPV16 is the one most commonly associated with this cancer.

  Human papillomavirus infection is also associated with cervical cancer precursor lesions and squamous intraepithelial lesions. While most low-grade squamous intraepithelial lesions spontaneously disappear in the future, some progress to advanced squamous intraepithelial lesions. These advanced lesions, particularly cervical squamous intraepithelial neoplasia 3, are associated with a high rate of progression to invasive cervical cancer.

  Two early gene products, E6 and E7, mediate transformation to a malignant phenotype by human papillomavirus. Both of these viral proteins have been shown to interact with the products of cellular human tumor suppressor genes. The E6 protein binds to cell-encoded p53 and can promote its degradation, while the E7 protein interacts with the retinoblastoma susceptibility gene product. HPV E6 / E7 protein constitutive expression is required to maintain the malignant phenotype of cervical cancer.

  Cellular immunity plays an important role in the control of human papillomavirus infection and human papillomavirus-related diseases. It is important for CD4 T cells to cause an antitumor response. The effectiveness of these CD4 T cells is believed to be in their ability to promote the priming and maintenance of CD8 cytotoxic T lymphocytes that are thought to act as dominant effector cells in tumor elimination. Immunohistochemical analysis of squamous intraepithelial lesions and cervical cancer specimen samples indicate that activated cytotoxic T lymphocytes are present in the lesions. CD4 T cells activate cytotoxic T lymphocytes by producing T helper 1 cytokines and providing an activation signal to prime tumor-specific cytotoxic T lymphocytes to professional antigen presenting cells To do. CD8 positive cytotoxic T lymphocytes recognize foreign peptides of 8-11 amino acids in length, bind to and are presented by human leukocyte antigen class I molecules. These peptides are called T cell epitopes.

  Memory T cells play an important role in maintaining long-term immunity against previously encountered pathogens or tumor antigens. These memory T cells can proliferate and rapidly acquire effector function, kill virus-infected cells or tumor cells, and secrete cytokines that inhibit pathogen replication after restimulation by re-exposure to antigen. Antigen-presenting cells that can transmit peripheral antigenic signals to lymphoid organs play an important role in inducing antigen-specific T cell immune responses against human papillomavirus infections and human papillomavirus-related tumors. Dendritic cells as professional antigen presenting cells express high levels of major histocompatibility complex and costimulatory molecules. Insufficient or inappropriate activation of dendritic cells leading to antigen presentation in the absence of proper costimulation caused by lack of pro-inflammatory signals is one reason for impaired anti-tumor immunity is there.

  Prophylactic HPV vaccines are available and work by preventing HPV infection. However, these prophylactic HPV vaccines are not effective in individuals who are already infected. Since standard surgical treatment is associated with an increased risk of preterm birth, HPV treatment vaccines will benefit women who have pre-cancerous lesions but wish to have children. It will also benefit men and women who live in developing regions of the world and do not have access to surgical modalities.

  Pharmaceutical formulations containing HPV peptides for use as therapeutic vaccines are provided. Also provided are methods for preparing formulations, particularly methods for dissolving sparingly soluble HPV peptides. Also provided are methods for treating HPV-related lesions, including HPV infections and HPV-related cancers.

  One embodiment comprises an HPV E6 peptide A comprising at least 20 contiguous residues of HPV E6 81-115 (residues 81-115 of SEQ ID NO: 1) of 20-100 amino acids in length. Prior to the step of dissolving peptide A, at least 50% HPV E61-80 (residues 1-80 of SEQ ID NO: 1) and at least 50% of each 10-100 amino acids in length HPV E6 116-158 (residues 116-158 of SEQ ID NO: 1) collectively containing two or more HPV peptides Y dissolved in a buffer, peptide A in dissolved form, peptide Y A method of dissolving HPV E6 peptide comprising creating a final soluble composition comprising in dissolved form. Peptide Y in the buffer prior to the step of dissolving peptide A is preferably in fully dissolved form (no insoluble peptide Y), and in the final soluble composition, peptides A and Y are preferably in fully dissolved form.

  Another embodiment is: (a) one or more HPV E6 peptides, each of 10-100 amino acid residues in length, (b) glutamic acid (salt) at a concentration of 2-40 mM, (c) 0 A drug having a pH of 3.0 to 5.0, comprising trehalose at a concentration of 3% to 5% (weight / volume), (d) glycine at a concentration of 0.2% to 10% (weight / volume) A formulation is provided.

  Another embodiment provides a pharmaceutical formulation comprising HPV E6 peptide A and one or more HPV peptides Y, the composition comprising HPV E6 81-115 (SEQ ID NO: 20-100 amino acids in length) HPV E6 peptide A comprising at least 20 contiguous residues of one residue 81-115), before the step of dissolving said HPV peptide A, at least 10-100 amino acids in length 2 collectively comprising 50% HPV E6 1-80 (residues 1-80 of SEQ ID NO: 1) and at least 50% HPV E6 116-158 (residues 116-158 of SEQ ID NO: 1) The above HPV peptide Y is dissolved in a buffer containing dissolved form, and the final soluble composition containing the peptide A in dissolved form and containing the peptide Y in dissolved form is created. It is produced by the method containing these.

  Another embodiment is: (a) one or more HPV E6 peptides, each of 10-100 amino acid residues in length, (b) glutamic acid (salt) at a concentration of 2-40 mM (c) 0.3 A human human papillomavirus (HPV) comprising administering a pharmaceutical formulation comprising trehalose at a concentration of 5% to 5% (w / v), (d) glycine at a concentration of 0.2% to 10% (w / v). ) To reduce infection or increase the regression of HPV-related lesions in HPV positive individuals.

  Herein, it is shown in Example 2 that a recall antigen such as CANDIN enhances the T cell immune response against the HPV peptide tested. The mixture of recall antigen and HPV peptide comes into contact with peripheral blood mononuclear cells. Thus, administering a vaccine comprising a recall antigen together with a disease specific antigen has general applicability and may promote a cellular (T cell) immune response against the disease specific antigen.

  Accordingly, one embodiment provides a method for inducing a T cell response to HPV by reducing infection by an individual's human papillomavirus (HPV) or increasing HPV-related lesion regression in an HPV positive individual, the method Administering to the individual a composition comprising one or more HPV antigens and administering to the individual a recall antigen that is not an HPV antigen, wherein the recall antigen is administered and contacted with the one or more HPV antigens of the individual. The individual needs a T cell response to one or more HPV antigens, and the one or more HPV antigens are not E6 antigens.

In a phase I trial of a patient with a woman with advanced squamous epithelium (HSIL) diagnosed by biopsy, women were HPV protein E6 residues 1-45 (SEQ ID NO: 2), E6 46-80 (SEQ ID NO: 2). 3), including E6 81-115 (SEQ ID NO: 4), and E6 116-158 (SEQ ID NO: 5), all treated with an intradermal injection of the composition mixed with candine as an adjuvant. The test dose was 50 ug, 100 ug and 250 ug of each peptide. Surprisingly, 4 out of 6 subjects in the 50 ug dose group (67%), 3 out of 6 subjects in the 100 ug dose group (50%), 3 subjects in the 250 ug dose group 0 of them had complete regression of their lesions. In addition, one additional subject in the 50 ug dose group had partial regression (remaining <0.2 mm ² lesions). This is a surprising result that the lowest dose was most effective. This is reported in Example 3 below.

  Thus, another embodiment consists of 25-110 ug of SEQ ID NO: 2 peptide, 25-110 ug of SEQ ID NO: 3 peptide, 25-110 ug of SEQ ID NO: 4 peptide, 25-110 ug of SEQ ID NO: 5 A unit dose pharmaceutical composition comprising a peptide and a recall antigen in an amount of 100-900 ul in a unit dosage form for intradermal injection is provided.

  Another embodiment is a peptide consisting of 25-110 ug of SEQ ID NO: 2, 25-110 ug of SEQ ID NO: 3, 25-110 ug of SEQ ID NO: 4, 25-110 ug of SEQ ID NO: 5, And a method of treating HPV infection comprising injecting into a patient a unit dosage pharmaceutical composition comprising the recall antigen in a unit dosage form for intradermal injection in an amount of 100-900 ul.

  Another embodiment includes intradermally administering to the subject a composition comprising one or more antigens of a microorganism and administering a recall antigen that is not a microbial antigen to the subject, wherein the recall antigen is administered. To provide a method of treating a disease caused by a microorganism in a mammalian subject that is in contact with one or more antigens of the subject microorganism.

Surface expression of CD1a (top), Langerin (middle), and E-cadherin (bottom) indicates successful conversion to LC (solid line). The dotted line indicates the relevant isotype control. Same as above. Same as above. LC maturation effect examined by surface expression of CD40, CD80, CD86, and HLA-DR. (A) Representative FACS histogram of subject 2. The shaded gray area, black dotted line, black solid line, short dashed line, and long dashed line indicate the isotype control, medium, candin, “peptide”, and candin / “peptide”, respectively. (B) Summary of results of all test subjects Same as above. LCs pulsed with T cell proliferation candies and candin / "peptides" measured using Alamar Blue induce a significant increase in T cell proliferation compared to media. All wells contained CD3T cells (1.5 × 10 ⁵ cells) and autologous LC (3 × 10 ³ cells). Shown are representative results of cytokine expression by LC of subject 4 treated with candin (150 μl / ml) or candin / “peptide”. Bars represent the standard deviation of the iterations. Intracellular cytokine staining of IFN-γ, IL-4 and IL-17A of CD4 T cells stimulated with LC pulsed with candin or candin / “peptide”. (A) Representative dot plot showing gating of subject 1 lymphocytes. (B) Representative dot plot of living cells of subject 1 sorted using eFluor450. (C) Representative dot plot showing IL-4 selected CD4 cells of subject 1 exposed to LC pulsed with candin / "peptide". (D) Corresponding isotype control of IL-4. (E) Representative dot plot showing IFN-γ selected CD4 cells of subject 1 exposed to LC pulsed with candin / “peptide”. (F) Corresponding isotype control of IFN-γ. (G) Representative dot plot showing IL-17A selected CD4 cells exposed to LC pulsed with candin / "peptide". (H) Corresponding isotype control of IL-17A. (I) A diagram summarizing the results of all subjects. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. FIG. 6 shows a diagram of the vaccination plan. Each subject will receive the same dose at all doses. CRC, Clinical Research Center; Colpo, Colposcopy; Bx, Biopsy FIG. 7 shows a dose escalation schedule for vaccination. A total of 24 subjects entered the dose escalation phase and an additional 30 subjects were recruited at the final dose. HPV16 E6 and E7 specific CD3 T cell responses before vaccination, after 2 vaccinations and after 4 vaccinations. T cell lines were established by stimulating CD3 T cells with autologous dendritic cells pulsed with HPV16 E6-vac, E6-GST, E7-vac, and E7-GST. Samples from separate visits were tested in the same ELISPOT assay using overlapping peptides, with each region tested in triplicate. Results for subjects showing a statistically significant increase relative to the E6 peptide are shown, and regions with a significant increase determined using the paired t-test are marked with “*”. Also in subject 11, HPV16 was detected before and after vaccination, and there was a significant increase relative to E7, possibly marked as “e”, which could represent the first example of epitope expansion. Circulating immune cells of vaccinated persons (n = 14) before vaccination, after 2 vaccinations and after 4 vaccinations. The percentage of lymphocytes is shown for CD4 cells. The percentage of CD4 and Tbet positive CD4 cells is shown for Th1 cells, the percentage of CD4 and GATA3 positive CD4 cells is for Th2 cells, and the percentage of CD4, CD25 and FoxP3 positive CD4 cells is for Treg. The percentage of monocytes positive for CD14 and HLA-DRlow / neg is shown for MDSC. Compared to before vaccination, the percent percentage of CD4 cells was significantly reduced after 4 vaccinations (paired t-test, p = 0.02). Bars represent the standard error of the mean. Schematic of the study visit scheduled for our Phase II clinical trial of HPV therapeutic vaccine. The blood test is for clinical analysis. Blood collection is for scientific analysis. CRSC, Clinical Research Services Core Unit; Colpo, colposcopy, Bx, biopsy, ECC, cervical intimal curettage, LEEP, electrosurgical loop resection

  The present invention relates to HPV peptides for use in therapeutic vaccines.

  Transformation of the squamous epithelium to a malignant phenotype by human papillomavirus is mediated by two early gene products—E6 and E7. Both viral proteins have been shown to interact with the products of cellular human tumor suppressor genes. The E6 protein binds to cell-encoded p53 and can promote its degradation, while the E7 protein interacts with the retinoblastoma susceptibility gene product. Expression of E6 and E7 open reading frames has been shown to be necessary and sufficient for transformation of human cells with HPV16.

  We have previously studied the epitopes of E6 and E7 that are recognized in the favorable immune response to HPV (Nakagawa, M. et al, 2010, Journal of Lower Genital Tract Disease, Vol. 14, No. 2, p. 124-129; U.S. Patent Publication Nos. 20110293651, 20090136531, 200901117140, and 2006018763).

  We have identified HPV E6 and E7 peptides for use in therapeutic vaccines, in particular HPV E6 peptides (US 20110293651, 20090136531, 200901117140).

  There are numerous types of HPV. The one most commonly associated with cancer is HPV16.

  The peptides described herein are derived from the HPV E6 protein (HPV E6). The sequence of E6 derived from HPV16 is SEQ ID NO: 1.

  The peptides of the following embodiments are HPV E6 peptides, meaning that they are derived from the sequence of HPV E6 protein. The E6 protein can be derived from any HPV strain. In a preferred embodiment, the peptide is derived from E6 of HPV16.

  Preferably the peptide comprises only the HPV E6 sequence. However, these peptides may contain other amino acid residues. These peptides may include E6 sequences from any HPV strain, not just HPV16.

  The peptides are preferably chemically synthesized, but they may be made in recombinant organisms by recombinant DNA technology. These peptides may also be made by other means known to those skilled in the art, for example by proteolysis of E6 or proteolysis of peptide peptides longer than the peptide to be produced.

  In some embodiments, the peptide is acetylated at its amino terminus, amidated at its carboxy terminus, or both. In other embodiments, neither end is modified.

  In certain examples, the peptides are 10-100, 8-100, 8-75, 8-50, 8-40, 10-75, 10-50, 10-40 in length. , 20-100, 20-75, 20-50, 20-40, 30-100, 30-75, 30-50, or 30-40 amino acid residues.

  In general, a peptide is “forward L” which has the sequence described and means that the amino acid is the L stereoisomer. However, in certain embodiments, the peptide can be a reverse D peptide, in which the ordinary sequence of amino acid residues is inverted, meaning that the amino acid is a D stereoisomer.

  One embodiment comprises an HPV E6 peptide A comprising at least 20 contiguous residues of HPV E6 81-115 (residues 81-115 of SEQ ID NO: 1) of 20-100 amino acids in length. Prior to the step of dissolving peptide A, at least 50% HPV E61-80 (residues 1-80 of SEQ ID NO: 1) and at least 50% of each 10-100 amino acids in length Dissolving two or more HPV peptides Y in a completely dissolved form, including peptide A in a completely dissolved form, collectively containing HPV E6 116-158 (residues 116-158 of SEQ ID NO: 1); A method of dissolving HPV E6 peptide comprising creating a final soluble composition comprising peptide Y in fully dissolved form.

  In certain embodiments, peptide A is acetylated at its amino terminus and amidated at its carboxyl terminus.

  In certain embodiments, HPV peptide A comprises residues 81-115 of SEQ ID NO: 1. In other embodiments, HPV peptide A comprises 25 consecutive residues of residues 81-115 of SEQ ID NO: 1 or 30 consecutive residues of residues 81-115 of SEQ ID NO: 1. .

  In certain embodiments, HPV peptide A consists of residues 81-115 of SEQ ID NO: 1.

  In certain embodiments, peptide A is acetylated at its amino terminus and amidated at its carboxyl terminus.

  In certain embodiments, the buffer has a pH of about pH 3.0 to about pH 5.0, about pH 3.5 to about pH 4.5, or about pH 2.5 to about pH 5.5.

  In certain embodiments, the buffer comprises at least 2 mM glutamic acid (salt). In other embodiments, the buffer comprises 2-50 mM glutamic acid (salt), at least 5 mM glutamic acid (salt), 5-50 mM glutamic acid (salt), or 5-25 mM glutamic acid (salt), or 2-25 mM glutamic acid (salt). Can have. The term “glutamic acid (salt)” in this context is intended to include all protonated and deprotonated forms of glutamic acid, namely glutamate and glutamic acid.

  In certain embodiments, peptides A and Y collectively comprise all of SEQ ID NO: 1 or all of HPV E6 sequences.

  In certain embodiments, peptide A consists of residues 81-115 of SEQ ID NO: 1 and peptide Y consists of three peptides consisting of residues 1-45, 46-80, and 116-158 of SEQ ID NO: 1. It is.

  In a more particular embodiment of the invention, each of peptides A and Y is acetylated at its amino terminus, amidated at its carboxyl terminus, and the buffer has a pH of about pH 3.0 to pH 5.0, After lysis, each of peptide A and three peptides Y has a concentration of 0.1-20 mg / ml. In other embodiments, after lysis, each of peptide A and three peptides Y has a concentration of 0.1-5 mg / ml or 0.02-5 mg / ml.

  In certain embodiments, each of peptide Y is a weight / volume concentration of at least 80% of peptide A in the final soluble composition.

  In certain embodiments, each of peptide A and peptide Y is 0.1-5 mg / ml in the final soluble composition. In other embodiments, each of the peptides A and Y is 0.1-20 mg / ml or 0.02-5 mg / ml.

  One embodiment is: (a) one or more HPV E6 peptides each of 10-100 amino acid residues in length, (b) glutamic acid (salt) at a concentration of 2-40 mM, (c) a concentration of A pharmaceutical composition comprising 0.3% to 5% (w / v) trehalose, (d) a glycine concentration of 0.2% to 10% (w / v), the composition having a pH of It is 3.0-5.0.

  Other possible ranges of glutamic acid (salt) concentration are 2-20 mM and 5-20 mM. Other possible ranges of trehalose concentrations are 0.2% to 5% (weight / volume), 0.5% to 5% (weight / volume), and 0.3% to 2% (weight / volume) ), And 0.5% to 2% (weight / volume). Other possible ranges of glycine concentration are 0.2% or more, 0.3% or more, 0.5% or more, 1% or more, and at most 3%, at most 5%, at most 8%, 10% at the maximum, 15% at the maximum, and 20% at the maximum.

  In certain embodiments, at least one of the one or more HPV E6 peptides comprises residues 46-70 of SEQ ID NO: 1, or comprises residues 91-115 of SEQ ID NO: 1, or SEQ ID NO: 1 Of residues 80-88. In certain embodiments, at least one of the one or more HPV E6 peptides comprises residues 46-70 of SEQ ID NO: 1 or comprises residues 91-115 of SEQ ID NO: 1.

  In certain embodiments, the pharmaceutical composition comprises at least 3 HPV E6 peptides, each comprising 10-100 amino acid residues, collectively comprising at least 50% of the HPV E6 sequence.

  In certain embodiments, the composition comprises at least one peptide consisting of residues 1-45, 46-80, 81-115, or 116-158 of SEQ ID NO: 1, residues 1-45, 46 of SEQ ID NO: 1. Comprising at least two peptides consisting of -80, 81-115, or 116-158, at least three peptides consisting of residues 1-45, 46-80, 81-115, or 116-158 of SEQ ID NO: 1, Or four peptides each consisting of residues 1-45, 46-80, 81-115, and 116-158 of SEQ ID NO: 1.

  In certain embodiments, each peptide is acetylated at its amino terminus and amidated at its carboxy terminus.

  The pharmaceutical composition can also include a recall antigen. Prototype recall antigens are commonly used in immunological skin tests to test immune responses, and in particular are epidemic parotitis antigens, Candida antigens, and ringworm fungus antigens. The test indicates whether the body “remembers” or “recalls” the antigen, ie, whether there is a delayed type hypersensitivity response in the skin where the antigen is administered by intradermal injection.

  The term “recall antigen” is defined herein as a substance or mixture containing a plurality of proteinaceous antigens, which is a mixture of most people who have been previously sensitized or exposed to the recall antigen in an intradermal skin test. Induces a delayed hypersensitivity response. Prototype recall antigens are commonly used in immunological skin tests to test immune responses, and in particular are epidemic parotitis antigens, Candida antigens, and ringworm fungus antigens. Each of these, referred to by the singular “antigen”, is actually composed of several or many molecular substances capable of inducing an immune response.

  In certain embodiments, the recall antigen can be an epidemic parotitis antigen (eg, inactivated whole mumps virus), Candida extract, or ringworm fungus extract.

  In certain embodiments, the recall antigen is an inactivated whole virus, an inactivated whole bacterium, or an inactivated whole microorganism.

  Example 2 below shows that Candida extract was involved in T cell proliferation in vitro in cells exposed to E6 peptide while E6 peptide had a partial maturation effect on Langerhans cells in vitro. Thus, Candida extract is an excellent adjuvant for the E6 peptide that induces a T cell response to potent HPV.

  We are conducting a trial on intradermal injection of four HPV E6 peptides and candin. The peptides were 10 mM glutamic acid (salt), 1.0% (weight / volume) trehalose, 2.0% (weight / volume) glycine, and 0.714 mg / ml of each of four HPV16 E6 peptides (SEQ ID NO: 1) A pharmaceutical solution comprising residues 1 to 45, 46 to 80, 81 to 115, and 116 to 158, each having its carboxy terminus amidated and its amino terminus acetylated) A. Drug solution A is sucked into the syringe in an amount of 50 μg, 100 μg, 250 μg, or 500 μg (70-700 μl of solution A) and mixed with 300 ul of candin in the syringe. The mixture in the syringe is then injected intradermally into HPV positive patients with cervical lesions.

Candine® (Candida albicans) is made from culture filtrates and cells of two strains of Candida albicans. The fungus grows in a medium with a known chemical composition consisting of an inorganic salt, biotin, and sucrose. The lyophilized raw material is extracted with a solution of 0.25% NaCl, 0.125% NaHCO ₃ and 50% (volume / deposition) glycerol. The concentrated extract is diluted with a solution of 0.5% NaCl, 0.25% NaHCO ₃ , 0.03% US Pharmacopeia albumin (human), 8 ppm polysorbate 80, and 0.4% phenol.

  The efficacy of Candine® (Candida albicans) is measured in humans by the DTH skin test. The procedure involves testing production lots simultaneously (in parallel) with an internal standard (IR) using sensitive adults previously screened and qualified to serve as test subjects. It is included. The caking reaction at 48 hours caused by the 0.1 mL production lot is measured and compared to the reaction caused by 0.1 mL IR. The test is within criteria if the potency of the production lot does not differ by more than ± 20% compared to the potency of IR when analyzed by a paired t-test (two-sided) with a p-value of 0.05.

  IR efficacy is monitored by the DTH skin test. People involved in the efficacy test are eligible for testing by taking four IR skin tests in which the mean induration response (mm) is calculated. The current skin test in IR has an IR potency greater than ± 20% compared to the mean eligible response in the same test subject when analyzed by a paired t-test (two-sided) with a p-value of 0.05 Show that it doesn't change. The caking reaction at 48 hours for the required IR is 15 mm ± 20%.

  The concentration of the Candine® (Candida albicans) skin test is determined from a dose response test in healthy adults. The product is intended to cause a caking reaction of ≧ 5 mm in immunologically qualified people with cellular hypersensitivity to the antigen.

  Another embodiment is: (a) one or more HPV E6 peptides, each of 10-100 amino acid residues in length, (b) glutamic acid (salt) at a concentration of 2-40 mM (c) 0.3 A human human papillomavirus (HPV) comprising administering a pharmaceutical formulation comprising trehalose at a concentration of 5% to 5% (w / v), (d) glycine at a concentration of 0.2% to 10% (w / v). ) To reduce infection or increase the regression of HPV-related lesions in HPV positive individuals.

  Another embodiment reduces administration of an individual with human papillomavirus (HPV) infection or regression of HPV-related lesions in an HPV-positive individual, comprising administering the pharmaceutical composition to an HPV-positive individual in need thereof Provide a way to increase In this case, the pharmaceutical composition may be a pharmaceutical composition comprising HPV E6 peptide A and one or more HPV peptides Y. The composition comprises 20 to 100 amino acids in length HPV E6 peptide A comprising at least 20 consecutive residues of HPV E6 81-115 (residues 81-115 of SEQ ID NO: 1). Prior to the step of dissolving peptide A, at least 50% HPV E61-80 (residues 1-80 of SEQ ID NO: 1) and at least 50% of each 10-100 amino acids in length Dissolving HPV E6 116-158 (residues 116-158 of SEQ ID NO: 1) collectively in a buffer containing two or more HPV peptides Y in dissolved form, comprising said peptide A in dissolved form, Made by a method comprising creating a final soluble composition containing peptide Y in dissolved form.

  In certain embodiments of these therapies, the method comprises injecting the pharmaceutical composition intradermally. The pharmaceutical composition may also be administered by other routes, such as intravenous or subcutaneous injection, or enter the intestine. However, intradermal injection is the preferred route.

  In certain embodiments of the therapy, the pharmaceutical composition further comprises a recall antigen.

  In certain embodiments of the treatment method, the method further comprises injecting a recall antigen intradermally.

  In certain embodiments, the method is a method of increasing the regression of HPV-related lesions in HPV positive individuals and the lesion is a malignant tumor.

  In certain embodiments, the lesion is cervical cancer.

  In certain embodiments, the lesion is head and neck cancer.

  In certain embodiments, the method is a method of increasing regression of HPV-related lesions, wherein the lesion is a tumor of the cervix, vulva, vagina, penis, anus, or pharynx.

  In certain embodiments, the method is a method of increasing regression of HPV-related lesions, wherein the lesion is a highly squamous intraepithelial lesion (HSIL).

  In other embodiments, the method is a method of increasing regression of HPV-related lesions in HPV positive individuals, wherein the lesion is a benign tumor or a precancerous lesion.

  The peptide in some embodiments is acetylated at its amino terminus, amidated at its carboxy terminus, or both. In other embodiments, neither end is modified.

    Preferably, in the method, the composition is administered by intradermal injection. However, the composition may be administered by any suitable method, eg, intramuscular injection.

  One embodiment provides a method of inducing a T cell response to HPV by reducing infection by an individual's human papillomavirus (HPV) or increasing HPV-related lesion regression in an HPV positive individual, the method comprising: Administering to the individual a composition comprising one or more HPV antigens and administering to the individual a recall antigen that is not an HPV antigen, wherein the recall antigen is administered and contacted with the one or more HPV antigens of the individual; An individual needs a T cell response to one or more HPV antigens. In certain embodiments, the one or more HPV antigens are E6 antigens or E7 antigens. In other specific examples, they are not E6 antigens. In another specific embodiment, they are not E7 antigens.

  The method does not involve administering a recall antigen that is not an HPV antigen, and is expected to produce a stronger T cell response to the HPV antigen in the administered individual compared to otherwise identical methods. A “stronger T cell response” is a subject treated with a mixture of a recall antigen and a disease specific antigen, eg, against a T cell from a subject treated with a disease specific antigen without a recall antigen in vitro. It can be shown by the greater cytotoxicity or antigen-specific T cell proliferative response mediated by antigen-specific T cells in the derived T cells. This is demonstrated by testing human subjects in clinical trials, more likely in animal model tests, or by in vitro testing of human-derived T cells, for example as shown in FIG. 3 of Example 2 below. be able to.

  Preferably, administration of one or more HPV antigens and recall antigens is performed by administering a composition comprising both one or more HPV antigens and recall antigens. However, the administration can also be a sequential separate administration of one or more HPV antigens and recall antigens, eg, intradermal injection of one or more HPV antigens in one composition and recall in a second composition. It can also be done by separate intradermal injections of the antigen at the same location.

  Thus, in one embodiment, a composition comprising one or more HPV antigens also includes a recall antigen.

  In one embodiment, administering the one or more HPV antigens to the individual and administering the recall antigen to the individual includes injecting the one or more HPV antigens and the recall antigen intradermally. In other specific examples, the recall antigen and HPV antigen are administered by subcutaneous injection. Langerhans cells are the most common antigen presenting cells in the skin and are found most abundantly, so intradermal injection is particularly preferred.

  In certain embodiments, the one or more HPV antigens comprises an HPV E7 antigen.

  In certain embodiments, the one or more HPV antigens are 8 to 100 amino acids, 8 to 70 amino acids, 8 to 50 amino acids, or a peptide of 8 to 40 amino acids in length. In a more specific example, one or more peptides are chemically synthesized.

In a phase I trial of a patient with a woman with advanced squamous epithelium (HSIL) diagnosed by biopsy, women were HPV protein E6 residues 1-45 (SEQ ID NO: 2), E6 46-80 (SEQ ID NO: 2). 3), including E6 81-115 (SEQ ID NO: 4), and E6 116-158 (SEQ ID NO: 5), all treated with an intradermal injection of the composition mixed with candine as an adjuvant. The test dose was 50 ug, 100 ug and 250 ug of each peptide. Surprisingly, 4 out of 6 subjects in the 50 ug dose group (67%), 3 out of 6 subjects in the 100 ug dose group (50%), 3 subjects in the 250 ug dose group Of these, 0 had complete HSIL regression of their lesions. In addition, one additional subject in the 50 ug dose group had partial regression (remaining <0.2 mm ² lesions). This is a surprising result that the lowest dose was most effective. This is reported in Example 3 below.

  Thus, another embodiment consists of 25-110 ug of SEQ ID NO: 2 peptide, 25-110 ug of SEQ ID NO: 3 peptide, 25-110 ug of SEQ ID NO: 4 peptide, 25-110 ug of SEQ ID NO: 5 A unit dosage pharmaceutical composition comprising a peptide and a recall antigen in an amount of 100-900 ul, 200-900 ul, 300-900 ul, or 100-600 ul in a unit dosage form for intradermal injection is provided.

  The recall antigen is sufficient to cause a caking reaction upon intradermal injection in humans and intradermal injections to the majority of immunocompetent adults who have previously been exposed to the antigen. And should be in concentration.

  In certain embodiments, the recall antigen is a Candida extract.

  In certain embodiments, the unit dose pharmaceutical composition comprises 200-400 ul of candine or equivalent total potency Candida extract.

  In certain embodiments of unit dose pharmaceutical compositions, the total amount is from 200 to 500 ul.

  In certain embodiments, the unit dose pharmaceutical composition comprises 30-70 ug of each peptide, or in other embodiments, about 50 ug of each peptide.

  In certain embodiments, each peptide is acetylated at its amino terminus and amidated at its carboxy terminus.

  In Example 3, injecting a composition containing 100 ug of each of the four peptides also worked well to cause lesion regression. Thus, another embodiment consists of 55-150 ug of peptide consisting of SEQ ID NO: 2, 55-150 ug of peptide consisting of SEQ ID NO: 3, 55-150 ug of peptide consisting of SEQ ID NO: 4, 55-150 ug of SEQ ID NO: 5 A unit dose pharmaceutical composition comprising a peptide and a recall antigen in an amount of 100-900 ul in a unit dosage form for intradermal injection is provided.

  Another embodiment comprises about 100 ug of a peptide consisting of SEQ ID NO: 2, about 100 ug of a peptide consisting of SEQ ID NO: 3, about 100 ug of a peptide consisting of SEQ ID NO: 4, about 100 ug of a peptide consisting of SEQ ID NO: 5, and a recall antigen. A unit dosage pharmaceutical composition comprising an amount of 100 to 900 ul in a unit dosage form for intradermal injection is provided.

  Another embodiment is to treat the patient with 25-110 ug of the peptide consisting of SEQ ID NO: 25, 25-110 ug of the peptide consisting of SEQ ID NO: 3, 25-110 ug of the peptide consisting of SEQ ID NO: 4, 25-110 ug of the SEQ ID NO: 5 There is provided a method of treating HPV infection comprising intradermally administering a unit dose pharmaceutical composition comprising a peptide comprising, and a recall antigen, in a unit dosage form for intradermal injection in an amount of 100-900 ul.

  In certain embodiments, the method comprises injecting the unit dose pharmaceutical composition into the patient at least three consecutive intradermal injections with no less than 5 days and no more than 28 days between each injection.

  In another embodiment, the method comprises injecting the unit dose pharmaceutical composition into the patient at least three consecutive intradermal injections with 10 to 21 days between each injection.

  In certain embodiments, the method comprises injecting the unit dose pharmaceutical composition into the patient intradermally at least twice consecutively, with 10 to 21 days between each injection.

  In certain embodiments, the method injects the unit dose pharmaceutical composition into the patient intradermally at least 3 times and at most 6 times within a period of 2 years, with 5 to 28 days between each injection. Including that.

  Example 2 herein shows that a recall antigen such as candin enhances the T cell immune response to the test HPV peptide. The mixture of recall antigen and HPV peptide comes into contact with peripheral blood mononuclear cells. Thus, administration of a vaccine containing a recall antigen together with a disease specific antigen has general applicability and may promote a cellular (T cell) immune response against the disease specific antigen.

  Thus, one embodiment includes administering to a subject a composition comprising one or more antigens of a microorganism and administering to the subject a recall antigen that is not a microorganism antigen, wherein the recall antigen is administered to the subject. A method of treating a disease caused by a microorganism in a mammalian subject in contact with one or more antigens of the microorganism.

  In certain embodiments, the microorganism can be a virus, bacterium, or fungus (eg, yeast). In certain embodiments, the microorganism is not HPV. In certain embodiments, the microorganism is not a herpes simplex virus.

  One or more antigens of the microorganism are 10-100, 8-100, 8-75, 8-50, 8-40, 10-75, 10-50, 10-40 in length Specific implementation of 2, 20-100, 20-75, 20-50, 20-40, 30-100, 30-75, 30-50, or 30-40 amino acid residues It can be a peptide in the example.

  The peptides are preferably chemically synthesized, but they may be made in recombinant organisms by recombinant DNA technology. The peptides may also be made by other means known to those skilled in the art, for example, by proteolysis of microbial proteins.

  The peptide in some embodiments is acetylated at its amino terminus, amidated at its carboxy terminus, or both. In other embodiments, neither end is modified.

  Preferably, in the method, the composition is administered by intradermal injection. However, the composition may be administered by any suitable method, eg, intramuscular injection.

Example 1
Dissolving the amidated and acetylated HPV E6 81-115 peptide and forming a pharmaceutical formulation We attempted to make a pharmaceutical formulation using four HPV E6 peptides. The four peptides were peptides consisting of residues 1-45, 46-80, 81-115, and 116-158 of SEQ ID NO: 1. Each peptide was amidated at its carboxyl terminus and acetylated at its amino terminus. Each peptide was chemically synthesized.

  The HPV16 E6 81-115 peptide was found to be insoluble in any suitable buffer for manufacturing. However, when added to a pH 4.0 solution of 10 mM glutamic acid already containing dissolved E61-45, E6 46-80, and E6 116-158, each of the four peptides had a concentration of 5 mg / ml. It was found that it was soluble and seemed to be dissolved.

  For pharmaceutical preparations, this was mixed with trehalose as a stabilizer and glycine as an osmotic pressure regulator. The final concentration of the formulation was 10 mM glutamic acid, 1.0% trehalose (w / v), 2.0% (w / v) glycine, and 0.714 mg / ml for each of the four peptides.

  The formulation was lyophilized for storage and redissolved by adding an appropriate amount of water for injection to obtain the above concentrations.

Example 2: Candida skin test reagents as novel adjuvants for therapeutic vaccines based on human papillomavirus peptides There is currently no vaccine adjuvant that can effectively promote cellular immunity. Because Candida skin test reagent injection has the ability to induce the resolution of common warts, our group is using it as a novel adjuvant in peptide-based human papillomavirus therapeutic vaccine trials. The purpose of this current study was to investigate the mechanism by which Candida enhances the vaccine immune response. Healthy subjects were examined for the maturation effect of Langerhans cells, the ability to proliferate T cells, the expression of cytokines and pattern recognition receptors by Langerhans cells, and the ability to induce Th1, Th2, and Th17 responses. Shows partial maturation effect on Langerhans cells suggested by significant upregulation of vaccine, human papillomavirus peptide with Candida, CD40 (p = 0.00007) and CD80 (p <0.00001) levels in vitro When presented by cells, it showed T cell proliferative capacity (p <0.00001). Interestingly, the maturation effect was attributed to the peptide, while Candida was responsible for T cell proliferation. Results of cytokine analysis of Langerhans cells treated with vaccine or Candida alone (IL-1β, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ, and TNF-α) are IL IL-12p70 protein was detected in the supernatant, indicating that -12p40 mRNA was induced with the highest frequency. Pattern recognition receptors known to be related to Candida albicans (DC-SIGN, Dectin-1, Dectin-2, Galectin-3, Mincle, Mannose receptor, Toll-like receptors-1, 2, 4, The presence of 6 and 9) was shown by all subjects. On the other hand, induction of a Th1 response indicated by IFN-γ secretion by CD4 cells stimulated with Langerhans cells pulsed with vaccine or Candida was shown in only one subject. In summary, the Langerhans cell maturation effect of the vaccine was attributed to the peptide, while the T cell proliferative capacity was derived from Candida, and the most frequently induced cytokine was IL-12.

Abbreviations: APC, antigen presenting cells; HPV, human papillomavirus; LC, Langerhans cells; MFI, mean fluorescence intensity; PAMP, pathogen-related molecular pattern; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; qRT-PCR, quantification Real-time PCR; PRR, pattern recognition receptor

1 INTRODUCTION The most widely used adjuvants for approved human vaccines are alum-based adjuvants that have been shown to elicit predominantly Th2 immune responses [1]. Thus, an alum-based adjuvant appears to be useful for vaccines designed to boost antibody responses, but not for vaccines designed to stimulate cellular immune responses. Since the successful elimination of human papillomavirus (HPV) infection is believed to be induced by cellular immunity [2, 3], an adjuvant that would promote such immunity is needed, but in hand I can't put it in.

  Our group and other groups included not only untreated wrinkles as well as the regression of warts treated by continuous intravaginal warts injection with skin test reagents such as Candida, Mumps, and / or Trichophyton. It has been shown that luxury regression can also be induced [4-9]. In a phase I trial (NCT00569231), our group treated common warts using Kandin® (Allermed, San Diego, Calif.), A colorless extract of Candida albicans. The resolution of the treated warts occurred in 82% of subjects, indicating an anti-HPVT cell response [8]. Given that candin is derived from Candida albicans, it should contain a number of pathogen-associated molecular patterns (PAMPs). We hypothesized that candin could be an effective vaccine adjuvant that would stimulate multiple pattern recognition receptors (PRRs) and induce innate and acquired immunity.

  Cervical cancer is most often caused by high-risk HPV infection and is the second most common cancer in women worldwide. Two highly effective prophylactic HPV vaccines, Gardasil® (Merck, New Jersey, USA) and Serricks® (GlaxoSmithKline, Middlesex, UK) are available These vaccines work by inducing high titers of neutralizing antibodies [10-12]. However, prophylactic HPV vaccines are not effective for women with pre-existing HPV infection [10, 12, 13]. Thus, there is no therapeutic HPV vaccine that can be used for people who are already infected with HPV and / or who have developed HPV-related tumors. Our group studies the naturally induced immunity in women with HPV infection and / or cervical lesions and the ability to induce a T cell response to E6, one of the high risk HPV oncoproteins Have been found to be associated with HPV exclusion and elimination of cervical lesions [3, 14, 15]. Therefore, we have designed an HPV therapeutic vaccine consisting of four HPV16 E6 peptides and Candin and are conducting a phase I trial (NCT016653249).

  In the current study, we examined the immune enhancement effect of candin as a vaccine adjuvant. Surprisingly, the E6 peptide was involved in the partial maturation of Langerhans cells (LC), while candin was involved in the T cell proliferative effect. The most induced cytokine by LC was IL-12.

2. Materials and Methods 2.1 Production of Monocyte-Derived LC Mononuclear cells were collected from healthy blood donors (n = 10) by apheresis (Key Biologies, LLC, Memphis, TN) . Subjects were numbered in chronological order. Peripheral blood mononuclear cells (PBMC) were purified using Ficoll gradient centrifugation. Monocytes are negatively isolated from PBMC using the monocyte isolation kit II (Miltenyi Biotech, Auburn, Calif.) And granulocyte / macrophage colony stimulating factor, IL− as described by Fahey et al. [17]. 4, and transforming growth factor β-1 was used to transform LC. The effectiveness of the conversion to LC has been demonstrated in selected experiments using CD1a (EBioscience, San Diego, CA), Langerin (Beckman Coulter, Blair, CA), and E-cadherin (EBioscience), FACS. Shown by detection using Fortessa (University of Arkansas for Medical Sciences Microbiology and Immunology Flow Cytometry Core Laboratory) and CellQuest Pro software (BD Biosciences, San Jose, Calif.) (FIG. 1). A sufficient number of cells could be obtained from all subjects except subject 1 for which the LC maturation experiment could not be performed.

2.2 Maturation analysis of LC treated with candin and / or HPV peptide Candin was dialyzed prior to use using a slide-A-riser G2 dialysis cassette (Thermo Scientific, Rockford, Ill.). A small amount of solvent (0.4% phenol) was removed. LCs were prepared as described above, and one million LCs were each candin (150 μl / ml), 4 current good manufacturing standard grade HPV16 E6 peptides [E6 1-45, E6 46-80, E6 81-115. , And E6 116-158 (hereinafter referred to as “peptide”); 10 μg / ml / peptide; manufactured by CPC Scientific, Sunnyvale, Calif., And vials by Integrity Bio, Camarillo, Calif. Or treated with candin / "peptide". A number of polysaccharide yeast cell wall particles [18] including zymosan (10 [mu] g / ml, In vivoGen, San Diego, CA), [beta] -glucan and mannan were used as positive controls. After 48 hours incubation, cells were stained with anti-human CD40 phycoerythrin (PE) -Cy5.5, CD80 fluorescein isothiocyanate, CD86PE-Cy5, and HLA-DR PE (EBioscience, San Diego, CA). Ten thousand events were acquired and data was analyzed using Flowjo software (BD Biosciences).

2.3 Analysis of T cell proliferation induced by LC treated with candin and / or “peptide” On the seventh day of LC transformation, the same subject CD3 T cells were transformed into pan T cell isolation kit II (Miltenyi Biotech). Was used to isolate negatively from PBMC. To remove CD25 regulatory T cells, human CD25 antibody-biotin (Miltenyi Biotech) was added. T cells (1.5 × 10 ⁶ ) in 100 μl complete Yssel medium (Gemini Bioproducts Inc, Woodland, Calif.) Containing 1% human serum in each well of a 96-well plate. T cell proliferation assay was performed in 6-well replicate wells by co-culturing cells / mL) with autologous LC (3 × 10 ⁴ cells / mL). Contains cells (T cells and LC) and contains cells and candin (150 μl / ml), cells and candin / “peptide”, and cells and tetanus toxoid (500 ng / ml, EMD Millipore, Billerica, Mass.) Wells were set up. After 7 days of incubation, 10 μl of Alamar Blue (Life Technologies, Grand Island, NY) was used to replace the volume corresponding to the medium in each well, and then the plate was incubated at 37 ° C. for 6 hours. Fluorescence was measured in culture using a BioTek Synergy-2 multiplate reader (US Biotech, Seattle, WA) (excitation wavelength 530 nm and emission wavelength 590 nm).

2.4 Cytokine and PRR analysis by quantitative real-time PCR (qRT-PCR) Candin (50 μl / ml, 100 μl / ml, and 150 μl / ml) with or without “peptide” (10 μg / ml / peptide) 1 million LCs were processed at each candin concentration. Zymosan was used as a positive control at 10 ug / ml and medium alone was used as a negative control. Cells were harvested for RNA after 8 and 24 hours. RNA was extracted using the RNeasy kit (Qiagen, Valencia, Calif.) And treated with DNase I (Promega, Madison, Wis.). cDNA synthesis was performed using the Superscript III first strand synthesis system (Life Technologies).

  Quantitative PCR analysis for cytokines including IL-1β, IL-6, IL-8, IL-10, IL-12p40, IL-23Ap19, IFN-γ, and TNF-α was performed using iQ-SYBR mix (Bio-Rad). , Hercules, Calif.). In addition, PRR known to be associated with Candida albicans (DC-SIGN, Dectin-1, Dectin-2, Galectin-3, Mincle, Mannose receptor, TLR-1, TLR-2, TLR-4) , TLR-6, and TLR-9) [19-28] were examined. The primers used to detect IL-12 have been previously reported by Vernal et al. [29]. All other primers were designed using Beacon Design software (Bio-Rad, Table 1). The threshold cycle number was normalized to the human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase and calculated as a fold change compared to untreated LC at 8 hours. . If cDNA amplification is indicated, it is considered that mRNA has been detected.

2.5 IL-12p70 protein analysis by ELISA The supernatant of LC treated with canine (50 μl / ml, 100 μl / ml, and 150 μl / ml) with or without “peptide” (10 μg / ml / peptide) At 24 hours, samples were taken from qRT-PCR experiments and tested using an IL-12p70 high sensitivity ELISA kit (EBioscience). The value of the medium only well is subtracted from the experimental well.

2.6 Intracellular cytokine staining The method was adapted from that described by Zielinski et al. [30]. CD4T cells were negatively isolated from PBMC using CD4T cell isolation kit II (Miltenyi Biotech) and co-cultured with autologous LC at a ratio of 50: 1 (CD4T cells: LC). Cells were stimulated with the addition of candin (150 μl / ml) with or without the “peptide” (10 μg / ml / peptide). Only medium was used as a negative control. Six days after co-culture, cells were stimulated with phorbol 12-myristate 13-acetate (200 nM, Sigma, St. Louis, MO) and ionomycin (1 μg / ml, Sigma) for 2 hours. Next, Brefeldin A (10 μg / ml, EBioscience) was further added for 2 hours. After staining with Fixable Viability Dye eFluor450 (registered trademark) (EBioscience), the cells were permeabilized / fixed, and anti-human IFN-γPE, IL-17A peridinin chlorophyll protein-Cy5.5, IL- Stained with 4 allophycocyanin or related isotype control (EBioscience). Ten thousand events were acquired using FACS Fortessa. Surviving lymphocytes were gated and the percentages of IFN-γ, IL-17A, and IL-4 positive CD4 T cells were analyzed using FACS Diva (BD Biosciences) and Flowjo software.

2.7 Statistical analysis Groups were compared using ANOVA with a mixed effects model, taking into account the dependency between groups. All paired comparison pairs were performed using Tukey's multiple comparison method for maturation markers (FIG. 2B), while medium control values were compared to the remaining groups using Dunnett's test for T cell proliferation (FIG. 3). .

3. Results 3.1 Phenotypic maturation of LC We evaluated the maturation effect of LC on Candin and / or “peptide” (FIGS. 1-2). For CD40, LC treated with zymosan (p <0.00001), “peptide” (p = 0.00003), and candin / “peptide” (p = 0.00007) compared to untreated LC Showed a statistically significant increase in mean fluorescence intensity (MFI). In addition, the MFI of LC treated with “peptide” and candin / “peptide” was significantly higher than that of LC treated with candin alone (p = 0.001 and 0.003, respectively). . For CD80, a significant increase in MFI was seen in LC treated with “peptide” (p <0.00001) and candin / “peptide” (p <0.00001) compared to media. Compared to LC treated with candin, CD80 expression was significantly higher in LC treated with “peptide” and candin / “peptide” (both p <0.00001). Only zymosan significantly increased the MFI of CD86 (p <0.00001). No significant increase was observed for HLA-DR. In summary, “Peptide” exerted a partial LC maturation effect while Candine did not. All endotoxin levels of “peptides” tested individually were below the limit of detection (<1.0 EU / mg).

3.2 T Cell Proliferation Measured with Alamar Blue Proliferation was significantly increased with candin (p <0.00001) and candin / “peptide” (p <0.00001) compared to the medium (FIG. 3). ). “Peptide” did not induce measurable proliferation. Measurable proliferation (> 5000 fluorescence increase) with tetanus toxoid was shown in subjects 2 and 5, but overall there was no significant increase compared to media (FIG. 3). It is unlikely that the possibility that LCs may have proliferated in addition to T cells cannot be ruled out.

3.3 Expression of cytokines by LC pulsed with candin or candin / "peptide" LCs of 10 subjects were treated with candin or candin / "peptide" and 8 cytokine mRNA expressions (Table 1) were obtained. It investigated by qRT-PCR (FIG. 4, Table 2). In selected experiments, the gel was purified and the amplification of the desired product was confirmed by DNA sequencing. Overall, the cytokine expression analysis results for LC treated with candin and candin / "peptide" were similar. IL-12p40 is the most frequently enhanced cytokine (> 5-fold compared to untreated) and its expression is 5 subjects for candin and 7 for candin / "peptide" Detected in subjects. IFN-γ was the second most frequently induced cytokine (6 subjects), detected in 5 subjects for candin and 4 subjects for candin / "peptide". IL-1β was also induced in 6 subjects, 4 subjects in Kandin and 6 subjects in Kandin / “Peptide”. IL-6 and IL-23p19 were induced only in candin (2 subjects for IL-6 and 1 subject for IL-23p19). TNF-α was expressed only in candin / “peptide” in one subject. IL-8 and IL-10 were not expressed in any subject.

  Supernatants from LC treated with candin or candin / "peptide" for 24 hours were analyzed for the presence of IL12p70 protein. IL12p70 is 27 out of 30 samples treated with candin (range 38-177 ng / ml) and 30 samples treated with candin / "peptide" (range 38-299 ng / ml). Of these, 27 were detected.

Table 1 Primers used for qRT-PCR

* TLR1 and 6 were analyzed by amplifying the 100% homology region between the two genes using the same primers.

Table 2
Summary of qRT-PCR results for the three most frequently increased cytokines

A magnification increase indicates ≧ 5.

3.4 LC PRR expression All eleven PRRs examined were detectable in the untreated LC of all subjects (data not shown). Few PRRs showed increased expression when stimulated with candin or candin / "peptide"(> 5-fold compared to untreated). No obvious difference was observed in the expression of PRR between LC treated with candin and LC treated with candin / "peptide". TLR-9 expression in 3 subjects (5-18 fold for candin, 9-16 fold for candin / "peptide") and Mincle expression in 2 subjects (candin and candin / " Peptide "5 times), Mannose receptor expression in 2 subjects (5-9 times in candin, 5-11 times in candin /" peptide "), Dectin-2 expression in 2 subjects (5 to 54 times for candin, 5 to 8 times for candin / "peptide"), as well as DC-SIGN expression increased in one subject (5 to 22 times for candin). In 5 subjects with increased PRR expression, 3 of them showed increased expression of 2 or more PRRs in LC.

3.5 Intracellular cytokine expression of CD4 T cells stimulated with LC pulsed with candin or LC pulsed with candin / "peptide" IFN-γ (Th1), IL-4 (Th2), and IL-17A (Th17 The CD4 T cells stimulated with LC treated with candin or candin / “peptide” were stained for 10 subjects (FIG. 5). In subject 4, an increase in IFN-γ secretion (> 5%) was seen in CD4T cells exposed to LC treated with candin or candin / “peptide” compared to the medium (9.5%, respectively). % And 6.9%). Overall, between CD4 T cells treated with LC alone and CD4 T cells treated with LC treated with candin, treated with LC treated with CD4 T cells alone and LC treated with candin / "peptide" There was no difference in the secretion of IFN-γ, IL-4, and IL-17A among CD4 T cells.

4 DISCUSSION “Adjuvant” comes from the Latin word adjuvale and means assisting or enhancing. An effective vaccine adjuvant should be able to promote a strong immune response against the vaccine antigen in terms of size and durability. Antigen presenting cells (APCs) play a crucial role in triggering immune responses. One desirable feature of an adjuvant is the ability to enhance APC maturation and the resulting priming of an effective T cell response. CD40 and CD80 have been shown to be critical for the activation of antigen-specific helper T cells [31] and cytotoxic T cells [32]. Our results show that “peptides” can induce significantly higher expression of CD40 and CD80. The present HPV therapeutic vaccine may be a rare vaccine in that it allows peptide antigens to mature APCs rather than adjuvants. These results differ from those reported by Romagnoli et al. [33], who showed upregulation of CD40, CD80, CD86, and HLA-DR on dendritic cells by Candida albicans. Since endotoxins are “peptides” and below the limit of detection, contamination is unlikely to contribute to the unexpected partial maturation effect on LC. Since our vaccine was formulated for the intradermal route to take advantage of sufficient LC in the epidermis, we focused on examining LC maturation effects. The study of maturation effects on other APCs such as dendritic cells and monocytes will be important in the future.

  Candida albicans as a component of normal flora often colonizes the skin and mucosal surfaces of healthy people. Potential acquired immunity against Candida albicans is usually present in individuals with normal immunity [34]. In this study, candin and candin / “peptide” induced significant T cell proliferation, but not “peptide”. Similar to our results, Gordon et al. [35] showed a positive skin test response to Candida albicans in 92% healthy subjects, Bauerle et al. Demonstrated a Candida-specific T cell response in 71% healthy subjects. Indicated. Candine has been used clinically to assess that cellular immunity is intact, and in this study we have shown that extracts from Candida albicans have a T cell proliferative effect. Consistent with finding. Unfortunately, however, the maturation effect of Candida albicans [33] is lost in the extract. On the other hand, it is found in this study that “peptides” exert some maturation effect.

  In the creation of this vaccine, since it is known that the E6 protein is hydrophobic, the obstacle faced was to allow the development of formulations in which the “peptide” was dissolved. While they remain dissolved at the acidic pH of the formulation, they remain insoluble and form microparticles at neutral pH (unpublished data). This unique property may contribute to the maturation effect by stimulating LC and phagocytosing these microparticles.

  PRR signaling can induce APC to express costimulatory molecules and cytokines necessary for T lymphocyte activation and differentiation [37]. Coordination of various PRRs with APC by stimulating multiple PRRs leads to synergistic Th1 responses [20, 38] and cytotoxic T lymphocyte responses [39]. Candida albicans is found in DC-SIGN [19], Dectin-1 [20], Dectin-2 [21], Galectin-3 [22], Mannose receptor [19], Mincle [40], and several TLRs It has been shown to activate many PRRs, including [25-27, 41, 42]. Since several PRRs increase during activation [43, 44], we examined the presence and amplification of these PRRs. In this study, all the PRRs studied were expressed by LC pulsed with candin and candin / "peptides", and certain PRRs (DC-SIGN, Dectin-2, Mincle, Monocyte Receptor, and TLR- The increased expression of 9) was shown in 5 out of 10 subjects. Further research is needed to determine which PRRs may be involved in signal transmission from the HPV therapeutic vaccine. Dectin-1 along with TLR-2 can activate NF-κΒ [20], and dectin-1 can independently mediate NFAT activation in dendritic cells and expression of inflammatory mediators such as IL-12p70 [45]. Therefore, it will be interesting in the future to investigate whether candin or candin / “peptide” plays a role in NF-κΒ and NFAT activation.

  Cytokines secreted by APC play an important role in the process of differentiating helper T cells into Th1, Th2 or Th17 cells. IL-12p70 induces a Th1 response, while IL-1β and IL-6 induce a Th17 response [37, 46]. The cytokine analysis results of the treated LC showed that IL-12p40 was the most frequently potentiated cytokine, and IL-12p70 was also detected at the protein level. Previous studies have shown that Candida albicans can induce differentiation of specific Th1 and Th17 cells [30, 33] and that a Candida-specific Th1 immune response can be detected in healthy subjects [47, 48]. From these data, we predicted that candine, an extract of Candida albicans, induces the skewing effect of Th1 and Th17. Although an increase in Th1 response (IFN-γ secretion> 5%) was observed in 1 subject, the overall results of 10 subjects showed no bias towards Th1 and Th17 responses. Candida may exert Th1 and Th17 effects through multiple mechanisms. There are other APC subsets in the skin, such as dermal DCs, that may play a role in the process of antigen presentation and T cell activation [49]. Furthermore, it will be important to evaluate the ability of the HPV therapeutic vaccines to induce HPV-specific T cell responses. This is being investigated in the ongoing clinical trial situation.

  In summary, “peptides” (antigens) are involved in LC maturation effects, while candin (adjuvant) induces significant T cell proliferation against the HPV therapeutic vaccine. Thus, antigens and adjuvants have complementary immune enhancing effects. Over time, ongoing clinical trials will reveal whether these complementary effects lead to an effective clinical response.

References for Example 2

Example 3 Phase I Trial of HPV Treatment Vaccine Containing E6 Peptide and Candin in a Woman with Advanced Squamous Intraepithelial Disease Diagnosed by Biopsy A Woman with Advanced Squamous Intraepithelial (HSIL) Diagnosed by Biopsy A single group, open-label, dose escalation phase I trial of an HPV therapeutic vaccine containing E6 peptide and candin in The vaccine is composed of a mixture of HPV peptide and candin. The peptides were 10 mM glutamic acid (salt), 1.0% (weight / volume) trehalose, 2.0% (weight / volume) glycine, and 0.714 mg / ml of each of four HPV16 E6 peptides (SEQ ID NO: 1) A pharmaceutical solution comprising residues 1 to 45, 46 to 80, 81 to 115, and 116 to 158, each having its carboxy terminus amidated and its amino terminus acetylated) A. Drug solution A is drawn into a syringe in an amount of 50 ug, 100 ug, 250 ug, or 500 ug (70-700 ul of solution A) and mixed with 300 ul of candin in the syringe. The mixture in the syringe is then injected intradermally into HPV positive patients with cervical lesions.

  Those who receive the vaccine are women with HSIL diagnosed by an untreated biopsy. Four vaccine injections (once every 3 weeks) are administered intradermally in the upper limbs. Before and after the second and fourth injections, blood is collected for CD3 ELISPOT and immunosuppressive cell analysis (to assess CD4 and CD8 responses). Clinical response is assessed by performing a LEEP resection after the fourth injection. An HPV-DNA test is performed before and after the fourth injection (FIG. 6). Each subject receives a single dose level for all four inoculations. Six subjects in the first cohort receive a 50 ug dose, and once that cohort is completed, the next subject receives the next higher dose level (details below and FIG. 7). After testing all doses (assuming no dose limiting toxicity), the maximum tolerated dose (MTD), immunological optimal dose (IOD), and clinical optimal dose (COD) are determined. An additional 30 subjects are vaccinated at the final dose (see below).

  Each of the first 6 subjects will receive a minimum dose (50 ug) of each peptide unless dose limiting toxicity is seen in 2 or more vaccination individuals. At each dose level, the first two subjects are staggered for at least one week as recommended by the FDA. As shown in FIG. 7, the dosage level is increased until the maximum tolerated dose is reached or the test is complete. Thirty additional subjects will be vaccinated at the final dose for further evaluation of clinical response.

  Simprep samples are tested for 37 HPV genotypes using the “Linear Array HPV Genotyping Test” (Roche Molecular Diagnostics, Alameda, Calif.) According to the manufacturer's instructions. The HPV types to be inspected are 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53. Type 54 type 55 type 56 type 58 type 59 type 61 type 62 type 64 type 66 type 67 type 68 type 69 type 70 type 71 type 72 type 73 type 81 type, 82 type, 83 type, 84 type, IS39 type, and CP6108 type. Human beta-globin signal was assayed as a positive control for sample validity of the DNA content of each sample. A positive control sample (including HPV plasmid DNA and the plasmid-encoded human beta-globin gene) and a negative control sample (no HPV plasmid DNA or human beta-globin gene) are supplied by the manufacturer and included in each experiment .

After each blood draw, PBMC are divided into CD14 ⁺ and CD14 ⁻ populations and stored frozen. To eliminate inter-assay variability, all three blood samples (before vaccination, after 2 vaccinations, and after 4 vaccinations) are used to establish T cell lines and perform ELISPOT assays. A CD3T cell line is established by stimulating CD3 cells magnetically selected in vitro with autologous mature dendritic cells exposed to HPV16 E6-vac, E7-vac, E6-GST, and E7-GST. Perform ELISPOT assay as described (1). 16 regions of HPV16 E6 and E7 proteins (E6 1-25, E6 16-40, E6 31-55, E6 46-70, E6 61-85, E6 76-100, E6 91-115, E6 106-130, E6 121-145, E6 136-158, E7 1-25, E7 16-40, E7 31-55, E7 46-70, E7 61-85, and E7 76-98). The assay is performed in triplicate. To compare each region before vaccination and after 2 vaccinations or after 4 vaccinations, a t-test of the corresponding specimen is performed as described above (2). Therefore, each subject is evaluated in terms of the number of regions with a statistically significant increase in T cell response after 2 or 4 inoculations.

For the measurement of circulating Treg cells and bone marrow-derived suppressor cells (MDSC), a small amount of PBMC (2 × 10 ⁶ cells) from each blood collection is used, and the level of circulating Treg and MDSC is monitored and vaccination is performed. Evaluate whether to inadvertently stimulate (3). CD4 ⁺ CD25 ⁺ forkhead box (FOX) P3 ⁺ cytotoxic T lymphocyte associated antigen 4 (CTLA-4) ⁺ number of cells, anti-human FoxP3 staining kit (Allophycocyanin, eBioscience, San Diego, CA) , CDLA-4 peridinin-chlorophyll-protein complex (BD PharMingen, San Jose, Calif.), CD25 phycoerythrin, and CD4 fluorescein isothiocyanate (BD Biosciences, San Jose, Calif.). (4). Cells are analyzed by flow cytometry (XL-MCL, Beckman Coulter, Fullerton, Calif.). The percentage of circulating Treg cells (% CD4 ⁺ CD25 ⁺ FoxP3 ⁺ CTLA-4 ⁺ / total CD4 ⁺ ) is determined before vaccination, after 2 vaccinations and after 4 vaccinations. If the proportion is at least twice as large after 2 or 4 inoculations as compared to before the inoculation, the Treg cells are increased. To count MDSCs, PBMC are stained with CD14 and HLA-DR antibodies and the percentage of CD14 ⁺ HLA-DR ⁻ / low is assessed (5). Representative cross sections of LEEP test samples are used for immunohistochemical staining with FOXP3 (rabbit polyclonal, Abcam, Cambridge, Mass.) To count the number of cervical Tregs (6). The density of FOXP3 ⁺ cells is determined using image analysis software and only cells with nuclear staining are counted.

result
Recruitment To date, 44 subjects have enrolled, 27 of whom have HSIL diagnosed by biopsy and have at least one vaccination (up to 27 additional subjects are 4/12) Vaccination before / 15).

Safety 99 injections were given to 27 subjects. > No grade 2 vaccine-related adverse events (AEs) have been reported (Table 3). (The two grade 3 events in Table 3 were not vaccine related.)
No grade 4 events have been reported.

Table 3

^a Appears at <24 hours from the time of vaccination; ^b Appears at> 24 hours from the time of vaccination; ^c > Feels hot in the absence of body temperature> 38.0 degrees

  The most common adverse events (AEs) were immediate injection site reaction and delayed injection site reaction (diffuse mild erythema at the injection site).

  In vitro studies have unexpectedly determined that the four current good manufacturing standards peptides targeting HPV16 E6 protein are up-regulated at CD40 (p = 0.00007) and CD80 (p <0.00001) levels Has been shown to have a maturation effect on Langerhans cells (LC) [30]. These maturation effects are likely due to the formation of microparticles (dissolved at the acidic pH of the formulation) by the peptide at neutral pH. Since insoluble microparticles are likely to be phagocytosed by LC, resulting in their activation and antigen presentation, the immediate and delayed injection site reactions seen during phase I trials are due to these microparticles. It can be a thing.

  Microparticles about 1-3 microns in diameter form when peptides dissolved at acidic pH are mixed with a buffer at neutral pH. Microparticles form whether or not the peptide is mixed with the Candida extract prior to addition to the neutral pH buffer.

Clinical response Results from 15 vaccinated individuals (mean age, 33.4 ± 6.5 years) can be used. Complete HSIL regression with 4 out of 6 subjects in the 50 ug dose group (67%), 3 out of 6 subjects in the 100 ug dose group (50%), 3 subjects in the 250 ug dose group It happened to 0 of them. Partial regression was defined as <0.2 mm ² HSIL lesions remaining at the end of study participation, one in the 50 ug dose group and one in the 250 ug dose group.

  The overall histological response rate was 60% (9 out of 15). This is higher than the regression rate of the historical placebo group in the range of 22% to 28%. Two of the subjects with HPV16 at the baseline of 5 (40%) had a regression, and 10 of the subjects with HPV other than 16 (7%) (70%) had a regression. None had progressed to squamous cell carcinoma.

Virus exclusion At least one HPV type could not be detected in 9 out of 15 subjects (60%). Of these 9 subjects, 7 (78%) showed a clinical response.

The response of CD3T cells induced by immune response vaccine to E6 (positive index ≧ 2) was detected in 10 out of 13 subjects (77%) and the increase was statistically significant in 6 subjects (46%) (Figure 8).

  After vaccination, the percentage of peripheral Th1 cells, Treg, and bone marrow-derived suppressor cells did not change, and the percentage of CD4 (p = 0.02) and Th2 cells decreased (FIG. 9).

In epithelium (1 mm ² per 127.4 ± 174.9 vs. 1 mm ² per 119.1 ± 115.6) and subepithelial stromal (1 mm ² per 351.9 ± 355 vs. 1 mm ² per 380.4 ± 152), The number of FoxP3-positive Tregs in non-responders (n = 7) lesions is different compared to responders (n = 7) lesions (CIN1 or representative normal areas) Not measured.

References for Example 3

Example 4 Need for Phase II Trial HPV Therapeutic Vaccines In the past decades, a number of preclinical trials and trials have evaluated prophylactic HPV vaccines, but these vaccines can be used in already affected HPV infected individuals. Is useless [51]. Gardasil, the tetravalent HPV L1 virus-like particle vaccine (HPV16, 18, 6, and 11) is the first, FDA approved in 2006, and three years later, the bivalent version (HPV16 and 18-type serverics were approved by the FDA. Trials have shown excellent vaccine efficacy in HPV 16 negative or HPV 18 negative women [52, 53], but the duration of protection has not yet been revealed. Also, bivalent vaccine trials show no evidence of increased viral clearance in women with existing HPV infection (n = 1,259; clearance is 35.5% in the vaccinated group, negative control vaccine) In the inoculated group, 31.5%, p = NS) [51]. Therefore, therapeutic vaccines are needed for cases already suffering from HPV infection and cases where HPV related diseases have already developed. This is especially true. This is because it has been reported that the target group (girl aged 13-17) has a preventive vaccination rate of only 32% nationwide [54]. Standard surgical treatments for HSIL, such as LEEP, are very effective [14], but have unintended side effects [14, 15] that increase the incidence of preterm birth from 4.4% to 8.9%. There has been concern. Since then, the latest guidelines no longer recommend treatment for CIN2 in young women (≤24 years in the narrow sense and any woman planning future pregnancy in the broad sense [14]). Treatment is still recommended for CIN3, but currently follow-up is considered acceptable. There is a real need for new therapies that do not change the anatomical integrity of the cervix, such as HPV therapeutic vaccines. In short, an HPV therapeutic vaccine is necessary because: (1) Prophylactic vaccines are not effective against already affected HPV infections, (2) Low use of prophylactic vaccines, (3) Therapeutic vaccines seem to leave the cervix intact (4) Therapeutic vaccines are other cancers caused by HPV such as anal cancer, pharyngeal oral cavity cancer, penile cancer, vaginal cancer, and vulvar cancer Can be effective against.

1.5.2 Rationale for Proposed HPV Peptide Dose In the Phase I trial, four dose levels (50, 100, 250, and 500 ug per peptide) were tested. The dose level with the highest clinical response will be selected and used in the Phase II trial. So far, a dose of 50 ug per peptide has a higher response rate (67% complete response and 17% partial response) compared to 100 ug per peptide (50% complete response).

  The initial four dose levels were chosen based on information available in the literature. Previous studies of trials with various peptide vaccines reported using doses ranging from 5 to 3,000 ug per peptide [31-38]. The optimal dose to achieve immunogenicity (and the lower dose when the two dose levels are the same) varied widely between vaccines: 96-mer malaria peptide 30 ug [31], prostate cancer 500 ug [34] of the therapeutic 9-mer peptide, 50 ug [35] of each of the 13 types of HPV16 E6 and E7 peptides ranging in length from 25 to 35 amino acids. Therefore, the dosage level that was likely to produce optimal immunogenicity was chosen.

  The clinically optimal dose from the four doses tested in the Phase I trial determined by the highest rate of histological regression (50, 100, 250, and 500 ug / peptide / injection) is It will be used as a phase trial dose.

1.5.3 Rationale for the proposed Candine® dose 300 μl of Candine® will be administered per injection. The amount is the amount used for intralesional injection of warts [47, 55] and the amount of candin used as a vaccine adjuvant in phase I trials. Since this amount has been shown to be safe and effective, the same amount will be used in Phase II trials.

1.5.4 Rationale for the proposed injection route The endothelial route will be used to utilize LC as antigen presenting cells. This route has also been shown to be safe, effective and immunogenic in Phase I trials and will be used in Phase II trials.

1.5.5 Rationale for Proposed Injection Sites Because of the ease of use and the availability of sufficient data showing the effectiveness of HPV peptides delivered from these sites, the extremities were chosen as the site of administration. [35, 56]. Limb injection has been shown to be safe, effective and immunogenic in Phase I trials and will be used for injections in Phase II trials.

1.5.7 Rationale for Injection Intervals In previous studies, the injection interval ranged from 2 weeks to 90 days [31-38], but the 3-week interval was mostly used. Kenter and colleagues found that if a blood sample was taken 7 days after the last vaccination, peptide vaccine immunogenicity as measured by the IFN-γ ELISPOT assay was rarely observed, but the blood sample was Reported that the immunogenicity was high when blood was collected 3 weeks after [35]. Therefore, we chose an interval of 3 weeks (± 7 days) as it seems long enough for the immune response to increase sufficiently. This interval has been shown to be safe, effective and immunogenic, and the same interval will be used for Phase II trials.

1.5.8 Rationale for the interval between the last injection and the final histological evaluation Although the histological response was assessed 3 months after the last vaccination by performing a LEEP in a phase I trial The complete effect is known to take a year [17-19]. In a phase II trial, PepCan will be administered as an alternative to LEEP, and histological response will be assessed by obtaining a colposcopy-induced biopsy 12 months after the last injection (Figure 10). In a trial treating high vulvular intraepithelial lesions using a similar peptide-based HPV therapeutic vaccine, histological regression increased by 25% to 47% between 3 and 12 months after vaccination [18].

1.5.9 Primary endpoint: Evidence rationale The clinical response to assess vaccine efficacy is based on punch biopsy results screening screening (eligible for vaccination with HSIL) and 12-month visit ( (± 2 weeks) to evaluate (FIG. 8). LEEP will not be used to assess efficacy, but will be provided free of charge to subjects with persistent HSIL at the 12-month visit.

  The proposed Phase II trial design is open-label, single center, and single group. We compare historical placebo groups from similar design trials (ie, enrollment of subjects with CIN2 / 3 diagnosed by biopsy and clinical response assessed by biopsy at 15 months) for comparison. It is planned to be used [57]. Of the first two dose levels tested in our Phase I trial, the 50 ug dose showed the best clinical response (4 of 6 or 67% complete response). Of the 117 people in the historical control group, 34 (29%) showed regression, so the 20 subjects in the vaccine group had 90% power (α = 0.05, tail = 2). It seems to be. However, the 67% clinical response rate is based on a small number of subjects. If we conservatively estimate the response rate is 55%, it would require 53 subjects (90% power, α = 0.05, tail = 2). Considering potential screening failures and trial withdrawal rates, we plan to screen 110 subjects and vaccinate 70 subjects in a phase II trial. While the use of the historic placebo group is not as rigorous as using the concurrent placebo group, the concurrent placebo group with CIN2 / 3 diagnosed by biopsy, which would remain untreated for 12 months, is ethically It seems difficult to justify.

1.5.10 Secondary endpoint: Safety rationale A mixture of HPV peptide and Kandin® was first tested in Phase I trials,> Grade 2 vaccine-related AEs reported It seems safe because there is nothing (Table 2). In phase II trials, CTCAE 4.03 will be used to assess safety as well.

1.5.11 Tertiary endpoint: Rationale for immune response and virus clearance 1.5.11.1 Rationale for measurement of HPV-specific T cell response HPV-specific CD3 T cell response is As done, before vaccination, after 2 vaccinations and after 4 vaccinations, it will be evaluated using an immunoassay such as the IFN-γ ELISPOT assay (FIG. 8). To assess the role of CD3T cells in vaccine efficacy, it will be assessed whether clinical response and viral clearance can be predicted based on CD3T cell activity.

1.5.11.2 Rationale for Measurement of Circulating Immune Cells Levels of circulating immune cells including CD4 T cells, Th1 cells, Th2 cells, regulatory T cells (Treg), and bone marrow derived suppressor cells (MDSC), It will be evaluated before vaccination, after 2 vaccinations and after 4 vaccinations. Preliminary data from a phase I trial shows that PepCan can reduce the Th2 response, resulting in increased effector immune activity (FIG. 9). These circulating immune cell levels will be used to determine if vaccine efficacy can be predicted in terms of clinical response and viral clearance.

1.5.11.3 Rationale for measurement of virus clearance HPV-DNA testing will be performed at the screening visit, the 6-month visit, and the 12-month visit (Figure 10). To date, all study participants have at least one type of HPV at the screening visit. The elimination of at least one type of HPV appears to correlate with clinical response. In Phase II trials, HPV types would be considered excluded if present at the screening visit but not at the 6-month and 12-month visits.

1.5.12 Rationale for other endpoints: predict vaccine response using a variety of factors such as age, HLA type, HPV type, proteomics analysis results, cytokine / chemokine analysis results, and laboratory tests; crossover Find protection and consider epitope expansion and cross-reaction as possible mechanisms. Not all vaccinees are expected to have a clinical response. Some people may have persistent HSIL, and others may progress to invasive squamous cell carcinoma. Identifying factors associated with a favorable response may be valuable, and as a result, knowledge-based decisions about who should respond to the vaccine and how long to wait before deciding to undergo surgical treatment Can do. Thus, employing a system biological approach, factors associated with clinical response and viral clearance can be determined.

  Phase I trials show that PepCan is effective against HSIL with HPV types 16 and HPV types other than 16. In Phase II trials, it can be determined for which types of HPV that are not type 16 PepCan is effective. Furthermore, epitope expansion and cross-reactivity can be examined as possible mechanisms behind cross-protection.

2 Objectives Main objective: Effectiveness 2.1
To assess the effectiveness of PepCan in a phase II trial by determining the clinical response that will be assessed by obtaining a colposcopy-guided biopsy at the 12-month visit. If a subject does not have any signs of CIN 2/3 during a 12-month visit biopsy, she will be considered a “responder”. It seems that some people retreat to CIN1, and some people do not have dysplasia. If there is still CIN 2 and / or 3 at the 12-month visit, the subject will be considered a “non-responder”. Secondary objective: Safety 2.2

  Safety will be assessed by recording AEs according to CTCAE v4.03 from the time of registration to the 12 month visit.

Tertiary purpose: immune response and virus elimination 2.3
Before vaccination, after 2 vaccinations, and after 4 vaccinations, immunological assessment in terms of HPV-specific CD3 T cell response would be assessed using the IFN-γ ELISPOT assay, , CD4, Th1, Th2, Treg, and MDSC cell circulating levels will be assessed by FACS analysis. Virological evaluation will be performed at the screening visit, 6-month visit, and 12-month visit.

Other purposes 2.4
To assess predictors for response to the vaccine (to determine which particular population of women should receive the vaccine and the timing of surgical treatment), age, HLA type, HPV type, proteomics analysis results, Cytokine / chemokine analysis results, clinical laboratory results, prophylactic HPV vaccination, smoking, oral contraceptive use, pap smear results, CIN grade (CIN2 vs CIN3), initial vital signs, body mass index, CD3T cell response to HPV16 E6, and Various parameters such as circulating immune cells can be analyzed.

  Cross-protection in terms of clinical response can be determined by counting each HPV event detected prior to vaccination in subjects exhibiting HSIL regression for each of the 36 HPV types tested (other than type 16).

  Cross protection from the point of virus clearance is present at each screening visit for each of the 36 HPV types tested, but by counting each HPV event that becomes undetectable at both the 6 and 12 month visits. Can be determined.

  Epitope expansion and cross-reactivity can be selected and examined in the subject.

3 Study drug test substance 3.1
3.1.1 HPV peptide PepCan has four HPV16 E6 peptides, namely E61-45 (Ac-MHQKRTAMMFQDPQER PRKLPQLCTELQTIHDIILECVYCKQQLL-NH2 (SEQ ID NO: 2)), E6 46-80 (Ac-RREVYDFAFRDCIDKY2AFRDVGD No. 3)), E6 81-115 (Ac-SEYRHYCYSLYGTTTLQQYNK PLCDLLIRCINCQK-NH2 (SEQ ID NO: 4)), and E6 116-158 (Ac-PLCPPEEKQRHLDKKQRFHNIRGRWT5) US No. 8,652,482). Commercially produced current good manufacturing practice (cGMP) grade peptides (CPC Scientific, San Jose, Calif.) Will be tested.

  Four peptides will be provided in lyophilized form in individual vials and stored at -70 ° C (acceptable range is -65 ° C to -75 ° C) except during transport and just prior to use. become.

3.1.2 Kandin (registered trademark)
The Candida albicans skin test antigen for cellular hypersensitivity will be supplied to the commercial drug Kandin (registered trademark). Vials will be stored at 2-8 ° C. as instructed in the package insert until use. This product is approved for multiple doses. The dose of Kandin (registered trademark) per injection in this test is 0.3 ml.

3.1.3 Mixing HPV Peptide and Candine® Sterile water will be added to a vial containing 4 current good manufacturing standards peptides. An appropriate amount of re-dissolved peptide will be drawn into a syringe depending on the dosage level, and 0.3 ml of Kandin® will be drawn into the same syringe. The mixed peptide-candin (R) mixture should be stored on ice or in the refrigerator until just prior to injection.

Treatment regimen 3.2
Subjects will receive 4 injections of PepCan (50-500 [mu] g / peptide / injection) and intradermal injections in the extremities with 3 weeks between each injection.

4 Outline of study design 4.1
This study is a single group of PepCan, an open-label, phase II trial for treating women with HSIL diagnosed by biopsy. The study design closely resembles current guidelines for treating young women with HSIL [14]. Study participants will be untreated patients with HSIL diagnosed by biopsy and patients referred from other clinics who attend a UAMS obstetrics and gynecology clinic. Four vaccinations (once every 3 weeks) will be administered intradermally to the limbs. Clinical response will be assessed by comparing colposcopy-induced biopsy results obtained before vaccination and at the 12-month visit. Safety will be monitored from enrollment through 12 months. For clinical testing and immunological analysis, blood will be collected before inoculation and after the second and fourth vaccination ("blood test"). To assist T cell analysis, blood will be collected after the first and third vaccinations and as much as possible at 6 months, 12 months, and at any LEEP visit ("Blood Collection"). HPV-DNA testing will be done at screening and at 6 and 12 months visits (Figure 10). If the subject has HSIL that persists at the 12-month visit, or if the subject ceases to participate in the study due to excessive toxicity, she will be given the option to return to the LEEP visit. Alternatively, she may choose to cease study participation and continue observation by the attending physician for up to two years as recommended before surgical treatment [14].

Toxicity monitoring 4.2
Severe toxicity will be defined (using CTCAE v4.03) as follows related to drugs.
Grade II or higher allergic reaction Grade II is “indication for therapeutic intervention or infusion interruption; responds immediately to symptomatic treatment (eg, antihistamines, NSAIDs, narcotic analgesics); indication for prophylactic medication for ≦ 24 hours Is defined as Grade III is “not responding quickly to long-term (eg, symptomatic drug treatment and / or short interruption of infusion); recurrence of symptoms following initial improvement; clinical sequelae (eg, kidney damage, Defined as "hospitalization adapted to lung infiltration".
Grade II or higher autoimmune response Grade II is defined as "indication of an autoimmune response involving non-essential organs or function (eg, hypothyroidism)". Grade III is defined as “autoimmune reaction involving major organs (eg, colitis, anemia, myocarditis, kidney)”.
• Any Grade III or higher event Any subject experiencing severe toxicity will be withdrawn from the study.

Cancellation rules 4.3
• If any subject experiences a grade IV or higher AE associated with the vaccine, study enrollment and vaccine administration will be suspended. These activities can only be resumed after permission has been granted by the Medical Monitor and applicable regulatory authorities.
• The sponsor may decide to quit the study at any point for any reason.

5 Target registration and test period 5.1. Target population, recruitment and informed consent process • Age 18-50 years, consulted at UAMS Obstetrics and Gynecology Clinic and ANGELS Telecolposcopy program, recent Pap smears are HSIL positive or “ Women who are the result of “Cannot rule out HSIL” will be recruited through physician referrals, booklets, flyers, UAMS websites, and word of mouth by the trial team. Potential subjects of interest will contact the study coordinator to discuss the study. The coordinator will perform an initial inclusion / exclusion criteria assessment, schedule the subject for a screening visit, and send a copy of the consent form for review by the subject.
・ Other women with recent abnormal pap smears that are positive for HSIL or “cannot exclude HSIL” have been referred to clinics, booklets, flyers (distributed inside and outside the university), UAMS websites, newspaper advertisements, radio, And / or will be recruited through social networking. Potential subjects of interest will contact the study coordinator to discuss the study. The coordinator will perform an initial inclusion / exclusion criteria assessment, schedule the subject for a screening visit, and send a copy of the consent form for review by the subject. The coordinator will ask the subject to obtain a copy of the pap smear results from the physician's office and bring it to the screening visit.
-Women who have recently been diagnosed with HSIL by colposcopy-guided punch biopsy (the period between the date of diagnosis and the first inoculation date must be ≤60 days), referral by clinic, booklet, flyer (university campus) Distributed outside), UAMS websites, and newspaper ads, radio, and / or social networking. Potential subjects of interest will contact the study coordinator to discuss the study. The coordinator will perform an initial inclusion / exclusion criteria assessment, schedule the subject for a screening visit, and send a copy of the consent form for review by the subject. The coordinator will ask the subject to obtain a copy of the medical record of the abnormal biopsy from the clinic and bring it to the screening visit.

5.1.1 Criteria for inclusion • Age 18-50 years • Recent pap smear results were consistent with HSIL or colposcopy-guided biopsy was “exclude HSIL” or HSIL・ HSIL or “HSIL can not be excluded” is untreated ・ Consent can be provided ・ I am willing or able to comply with the requirements of the protocol and can use English

5.1.2 Exclusion criteria • History or treatment history that causes immunosuppression (eg, cancer, HIV, organ transplantation, autoimmune disease) • Pregnancy or trying to become pregnant within study participation period • Study participation • Allergies to Candida antigen • History of severe asthma requiring emergency visits or hospitalization • Current use of beta-blocking medication (if anaphylaxis occurs) May not respond to epinephrine)
・ History of invasive squamous cell carcinoma of the cervix ・ If it is not the patient's best interest to participate in this study based on the opinion of the investigator or other investigator

5.1.3 Informed Consent Process • Potential candidates should receive the informed consent form prior to the screening visit and be given as much time as possible to decide on study participation. I will.
• Study coordinators / study team members who have been given the authority to provide informed consent will be discussed in detail with study candidates (announced periodically by the American Colposcopy and Cervical Pathology Society). Including age-specific criteria in the guidelines). At any time prior to the screening visit or at a screening visit, at the UAMS Gynecology Clinic (prior to any trial related procedures), all questions related to the trial that the subject may have will be answered. The explanation will be in English.
• Since outlets are an ongoing process, subjects will be asked whether they still want to participate in the study before the study procedure at each study visit.

Registration pace 5.2
During the phase I trial, approximately two-thirds of enrolled subjects were eligible for vaccination. Considering the screening failure rate and the test withdrawal rate (currently about 5% per year), we plan to enroll 110 subjects for screening and start vaccination for 70 subjects.

Test period 5.3
The test period will be up to 66 months. If a LEEP resection is performed, each subject is expected to be in the trial for about 16 months or longer.

6 Clinical trial visit scheduling 6.1
The study coordinator schedules the UAMS Obstetrics and Gynecology Clinic and Clinical Research Services Core (CRSC) study visits (screening, vaccination, 6 months, 12 months, and optional LEEP visits) Will. Screening, 6 months, 12 months, and voluntary LEEP visits are expected to take approximately 90 minutes. However, it may take longer on busy clinic days. The vaccination visit is expected to take about 60 minutes.

Acceptable period for clinical trials 6.2
6.2.1 Interval between visits to individual subjects • After the first vaccination visit (Visit 1), all screening visit results are available as much as possible and the subject is deemed eligible to continue with the vaccination phase of the study. , Should be scheduled within 60 days from the date the punch biopsy was obtained (screening date for most subjects).
• Subsequent vaccination / test (lab) visits (Visits 2-5) should be scheduled for 3 weeks ± 7 days.
• A 6-month visit should be scheduled 6 months + 2 weeks after the fourth visit.
• A 12-month visit should be scheduled 6 months + 2 weeks after the 6-month visit.
• Voluntary LEEP visits (if the subject chooses) should be scheduled as soon as possible after a 12-month visit or after the subject has ceased study participation due to severe toxicity.

Screening visit 6.4
6.4.1 Screening Visit Procedures • Reconfirm inclusion / exclusion criteria • Obtain consent form (if not already obtained) • Target contact information (Appendix 2) during visit・ Get the “Screening Visit Survey Form” (Appendix 3) to be filled in during the visit ・ Get demographic information ・ Get the subject's medical history
・ History: Be sure to ask about the history of the abnormal Pap smear so far and how it was treated.
・ Drug allergies
・ Concomitant medication ・ Physical examination
・ Get vital signs
Blood pressure (<200 / 120mmHg is acceptable)
・ Heart rate (50-120 bpm is acceptable)
・ Respiration rate (<25 bpm is acceptable)
Body temperature (<100.4 ° F)
・ Weight (no limit)
・ About subjects with the possibility of pregnancy
Explain the risks associated with pregnancy while receiving the vaccine
-Ask which contraceptive methods you are considering to use while participating in a vaccine trial. FDA approved forms include: sterilization, implantable rod, IUD, shot / injection, oral contraceptives, barrier method (vaginal ring, condom, pessary, cervical cap), And emergency contraception.
・ We perform colposcopy
・ Obtain a syrup for HPV-DNA testing
If a doctor deems necessary, obtain a punch biopsy and cervical intimal curettage (HSIL needs to be confirmed to be eligible)
If the lesion area cannot be viewed with colposcopy, the doctor may obtain a quadrantal blind biopsy
Record lesions, position on cervix, (if available) take cervical image using colposcopic image acquisition system, indicate where biopsy was taken
Record how many cervical quadrants can see the lesion
• If the subject has already been diagnosed with HSIL by biopsy, there is no need to do it again. However, colposcopy may be performed again to record the lesion site and take a symprep for HPV-DNA testing.
• Tube blood collection for CBC, liver function, and kidney function (performed in UAMS clinical laboratory)

Inoculation visit (Visit 1-5) 6.5
6.5.1 Procedures related to Visit 1-Ask whether any medication has been started or stopped since the last visit.-Pre-vaccination urine pregnancy test-Measure height and weight to determine BMI.-Vital sign before vaccination.・ We will collect blood for
Immunomonitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
CBC (one 3.0 ml purple cap EDTA tube, performed in UAMS clinical laboratory)
Liver panel and kidney panel (two 4.5 ml light green cap lithium heparin tubes, performed in UAMS clinical laboratory)
・ Vaccine injection ・ Vital sign at least 30 minutes after injection ・ Receive any immediate adverse reaction ・ Provide ibuprofen or naproxen ・ Subject Diary (included) Hand the document 4), fill out the subject, and ask to bring it with you at the next visit.

6.5.2 Procedures related to Visit 2-Ask for the completed "Target Diary". If the subject does not return it, ask "what have I experienced any side effects since the last vaccination"?
-Asking if any medication has started or stopped since the last visit-Prevaccination urine pregnancy test-Taking vital signs before vaccination-Blood will be collected for
• Immune monitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
・ Vaccine injection ・ Vital sign again after at least 30 minutes after inoculation ・ Monitor any immediate adverse reaction ・ Provide administration of ibuprofen or naproxen ・ Handing “diary” (Appendix 4), Fill out the subject and ask them to bring it on the next visit

6.5.3 Procedures related to Visit 3-Find the “Target Diary” you have entered. If the subject does not return it, ask "what have I experienced any side effects since the last vaccination"?
-Asking if any medication has started or stopped since the last visit-Prevaccination urine pregnancy test-Taking vital signs before vaccination-Blood will be collected for
• Immune monitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
CBC (one 3.0 ml purple cap EDTA tube, performed in UAMS clinical laboratory)
Liver panel and kidney panel (two 4.5 ml light green cap lithium heparin tubes, performed in UAMS clinical laboratory)
・ Vaccine injection ・ Vital sign again after at least 30 minutes after injection ・ Provide ibuprofen or naproxen administration ・ Monitor any immediate adverse reactions ・ Target diary (Appendix 4) Hand it over, fill in the subject, and ask to bring it with you at the next visit

6.5.4 Procedures related to Visit 4 ・ Find the “target diary” you have entered. If the subject does not return it, ask "what have I experienced any side effects since the last vaccination"?
-Asking if any medication has started or stopped since the last visit-Prevaccination urine pregnancy test-Taking vital signs before vaccination-Blood will be collected for
• Immune monitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
・ Vaccine injection ・ Vital sign again after at least 30 minutes after inoculation ・ Monitor any immediate adverse reaction ・ Provide administration of ibuprofen or naproxen ・ Handing “diary” (Appendix 4), Fill out the subject and ask them to bring it on the next visit

6.5.5 Procedures related to Visit 5-Ask for the “target diary” you have entered. If the subject does not return it, ask "what have I experienced any side effects since the last vaccination"?
-Blood will be collected for
• Immune monitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
・ CBC (one 3.0ml purple cap EDTA tube)
・ Liver panel and kidney panel (two 4.5ml light green cap lithium heparin tubes)

6 month visit 6.6
The 6 month visit will be scheduled approximately 6 months (± 2 weeks) after vaccination 4.

6.6.1 Procedure for 6-month visit ・ Ask if any medication has started or stopped since the last visit ・ Perform colposcopy
・ Obtain a syrup for HPV-DNA testing
-Record lesions, position on the cervix, (if available) take a cervical image using a colposcopic image acquisition system,
Record how many cervical quadrants can see the lesion
• Obtain a punch biopsy and cervical intimal curettage if deemed necessary by the physician (only if suspected of progressive disease) • Some subjects may further cross-react based on ELISPOT assay results , Epitope expansion, and / or characterization of novel T cell epitopes will be investigated. Blood collection will be performed
-6-8 10.0ml green rubber cap heparin sodium tubes

Visit for 12 months 6.7
The 12 month visit will be scheduled approximately 6 months (± 2 weeks) after the 6 month visit. 6.7.1 Procedure for 12-month visit ・ Perform physical examination
・ Get vital signs
·blood pressure
·Heart rate
・ Respiration rate
・ Body temperature
·body weight
-Ask whether any medication has started or stopped since the last visit.-Perform colposcopy.
・ Obtain a syrup for HPV-DNA testing
• Record lesions, position on the cervix, and take images of the cervix using a colposcopic image acquisition system (if available)
Record how many cervical quadrants can see the lesion
・ Punch biopsy and cervical intimal curettage as much as possible
・ If the doctor determines that it is necessary, perform cervical endometrial curettage ・ As explained above, blood may be collected from several subjects for the following reasons:
• Immune monitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
・ Please have the “12 Month Visit Survey Form” (Appendix 7) filled in during the visit.

6.7.2 12-month Visit Follow-up Study Coordinator and investigator or Co-Investigator will review all 12-month visit information and study results. If the biopsy shows no signs of HSIL, the subject will complete the study. If there is a persistent HSIL, the subject will choose to (1) have his or her gynecologist consult a further year before LEEP or (2) receive LEEP as part of the study It's okay.

Optional LEEP visit 6.8
6.8.1 LEEP Visit Procedures • As explained above, several subjects can be bled for the following:
• Immune monitoring and other analysis (6-8 10.0 ml green rubber cap heparin sodium tubes)
・ Perform LEEP biopsy
・ Obtain a sample preparation sample for HPV-DNA testing
Remove the visible lesion or if no visible lesion is seen, remove the biopsy from the area obtained at the 12-month visit

8 Evaluation items Clinical evaluation (UAMS clinical laboratory) 8.1
Clinical response is assessed (by a pathologist from the Department of Pathology) by comparing punch biopsy results from screening (having HSIL is an inclusion criterion) with punch biopsies performed at a 12-month visit become. A subject is considered a “responder” if the 12-month biopsy is HSIL negative (no signs of CIN 2/3), and is considered a “non-responder” if the biopsy shows HSIL (CIN 2 and / or 3) Will be.

Virological test HPV-DNA test (Nakagawa Laboratory) 8.2
Simprep samples will be examined for the presence or absence of HPV-DNA. Commercially available kits such as “Linear Array HPV Genotyping Test” (Roche Molecular Diagnostics, Alameda, Calif.) May be used. This kit consists of 37 HPV types (6 type, 11 type, 16 type, 18 type, 26 type, 31 type, 33 type, 35 type, 39 type, 40 type, 42 type, 45 type, 51 type, 52 type 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 Type 81, 82, 83, 84, IS39, and CP6108). The human beta-globin signal will also be assayed as a positive control for sample validity of the DNA content of each sample. A positive control sample (including HPV plasmid DNA and the plasmid-encoded human beta-globin gene) and a negative control sample (no HPV plasmid DNA or human beta-globin gene) are supplied by the manufacturer and included in each experiment It will be. HPV31 type, 33 type, 35 type, 52 type, 58 type, and 67 type are regarded as “HPV16 type related” and HPV18 type, 39 type, 45 type, 51 type, 53 type, 56 type, 59 type , 66 type, 68 type, 69 type, 70 type, 73 type, and 82 type are considered “high risk” and are 6 type, 11 type, 40 type, 42 type, 54 type, 61 type, 62 type, The 71, 72, 81, 83, 84, and CP6108 types will be considered “low risk” [58].

  Virological response will be assessed by comparing HPV-DNA test results before and after vaccination. A subject will be considered a “clearer” if at least one HPV type present before vaccination becomes undetectable at both the 6-month visit and the 12-month visit. Otherwise, the subject will be considered a “persistor” as long as it is detected at least one HPV type baseline.

Immunological evaluation 8.3
8.3.1 ELISPOT assay (Nakagawa Laboratory)
An immunoassay will be performed to assess the presence or absence of HPV specific T cells such as an ELISPOT assay. After each blood draw, PBMC will be separated into CD14 ⁺ and CD14 ⁻ populations and stored frozen. To eliminate inter-assay variability, all three blood samples (before vaccination, after 2 vaccinations, and after 4 vaccinations) should be used to establish T cell lines and perform ELISPOT assays become. Establishing a CD3T cell line by stimulating CD3 cells magnetically selected in vitro with autologous mature dendritic cells exposed to HPV16 E6-vac, E7-vac, E6-GST, and E7-GST become. The ELISPOT assay will be performed as previously described [28]. We typically have 16 regions within the HPV16 E6 and E7 proteins (E6 1-25, E6 16-40, E6 31-55, E6 46-70, E6 61-85, E6 76-100, E6 91-115. , E6 106-130, E6 121-145, E6 136-158, E7 1-25, E7 16-40, E7 31-55, E7 46-70, E7 61-85, and E7 76-98). If sufficient cells are available, the assay will be performed in triplicate. To compare each region before vaccination and after 2 vaccinations or after 4 vaccinations, the corresponding specimens will be t-tested as previously described [59]. Therefore, each subject was evaluated in terms of the number of regions with statistically significant increases determined using Student's paired t test after 2 or 4 inoculations. Will do. The remaining CD3 T cells may be used to assess recognition of homologous epitopes from other high-risk HPV types, describe new epitopes, and / or assess internal processing of such epitopes.

8.3.2 Measurement of immune cells 8.3.2.1 Circulating immune cells (Nakagawa Laboratory)
In addition, a small amount of PBMC (approximately 3 × 10 6 cells) from blood collected at Visit 1, Visit 3, and Visit 5 was used to monitor the levels of circulating immune cells such as Treg and MDSC, and vaccination It will be assessed whether the level of can be reduced [60]. Flow cytometry will be used to determine the number of CD4 + CD25 + FOXP3 + (Treg) and CD14 + HLA-DR− / low (MDSC) cells [29, 61]. Tbet (Th1), GATA3 (Th2), and / or ROR gamma T (TH17) positive cells may also be examined. The number of circulating immune cells will be determined before vaccination, after 2 vaccinations and after 4 vaccinations.

8.3.2.2 Cervical immune cells (UAMS Experimental Pathology Core)
After routine pathological diagnosis was made from a LEEP sample obtained at any LEEP visit, additional sections were made of CD3 (T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD56 (NK cells), CD1a (Langerhans cells important for antigen presentation), CD20 (B cells), CD68 (macrophages), FOXP3 (Treg), Tbet (Th1), and MadCAM-1 (addressin involved in T cell infiltration) are positive. Cervical immune cells may be examined. Eosinophils (Th2) may also be examined.

8.3.3 Other Additional analyzes that can be performed using blood samples to assess vaccine response include antibody production to HPV protein, cytokine response (Nakagawa Lab), and changes in protein expression (UAMS Proteomic Score Lab ( Proteomics Core Laboratory)).

9 Evaluation of data analysis effectiveness 9.1
Historical placebos from previously reported trials including similar trial designs (ie, enrollment of subjects with CIN 2/3 diagnosed by biopsy and clinical response assessed by biopsy at 15 months) Groups will be used for comparison [57]. Response rate of vaccinees who completed the trial (estimated to be 50-60 subjects) was 29% (34/117) of the historical placebo group and Fisher's exact test Will be compared. See “Theoretical Basis for Key Endpoints: Efficacy” (Section 1.5.9) for power analysis and justification for the number of subjects. Safety assessment: Summary of adverse events 9.2

  Subjects who have received at least one vaccine will be included in the safety assessment. The results will be tabulated as shown in Table 3. The type of adverse reaction, CTCAE grade, and whether the response is vaccine related will be indicated.

Assessment of immune response and viral clearance 9.3
9.3.1 Immune Response 9.3.1.1 CD3 T Cell Response to HPV As shown in FIG. 8, to compare each region before vaccination and after 2 or 4 vaccination, A t-test of a sample having a corresponding street will be performed.

  Correlation between CD3T cell response to HPV and clinical response was investigated by creating a contingency table for many subjects with at least one region with a statistically significant increase in E6 in “responders” and “non-responders” Will be. Fisher's exact test will be used.

9.3.1.2 Circulating immune cells As shown in FIG. 9, the percentage of circulating immune cells such as CD4, Th1, Th2, Treg, and MDSC after 2 and 4 vaccinations Changes will be compared at baseline. A paired t-test and one-way analysis of variance will be performed to determine statistical significance.

  The correlation between circulating immune cells and clinical response will be examined. The change in the percentage of circulating immune cells between the pre-vaccination and post-vaccination samples will be compared between “responders” and “non-responders”.

9.3.2 Virus Exclusion HPV-DNA testing will be performed using symptople samples from screening, 6-month, and 12-month visits.

  Correlation between CD3 T cell response to HPV and virological response is to create a contingency table for many subjects with at least one region with a statistically significant increase for E6 in “clearer” and “persistor” Will be considered. Fisher's exact test will be used.

  The correlation between circulating immune cells and viral clearance will be examined. The change in the percentage of circulating immune cells between the pre-vaccinated sample and the sample after 4 vaccinations will be compared between “clearer” and “persistor”.

Factors contributing to the recruitment and retention rate of study participants 9.4
Based on the data provided in the “screening visit questionnaire”, “early discontinuation questionnaire”, and “12-month visit questionnaire”, factors contributing to the target recruitment and retention can be evaluated. Using Fisher's exact test, factors such as Facebook's private group usage frequency, motivation to participate in the study, etc. will be compared. It will be compared between completed subjects.

Predictors of clinical response and viral clearance 9.5
Since proteomic data will be collected at three time points, we identify cluster clusters of proteins that are associated with specific dynamic responses to vaccines (eg, increase, decrease, U-shape) and Predicted protein expression characteristics will also be identified. Protein clustering was originally developed for gene expression microarray time series data, but has been successfully applied to proteomic data [63] using Mfuzz [62], a noise-robust clustering method become. We will analyze cluster clusters of test proteins for specific gene ontology (GO) annotations with high frequency to elucidate the root causes of different responses to vaccines. In addition to proteomics data, we have other varieties for predicting vaccine response, first by univariate analysis and then by multivariate analysis including variable selection using lasso [64] with 10-fold cross validation. The variable will be analyzed. Computer calculations will be performed in the R environment and the R / Bioconductor [65] environment. While variable selection using lasso will be performed using package glmmLasso, enrichment analysis of gene ontology function information (Gene Ontology terms) will be performed using topGO.

Definition 10.1
10.1.1 Adverse Event An adverse event is any occurrence or exacerbation of an undesirable or unintended sign, symptom, or disease that is temporally related to vaccine use, and is commonly used in the Common Terminology Criteria. for Adverse Events) (CTCAE) version 4.03 will be graded. Local and / or systemic adverse events may include itching, burning, pain, skin detachment, rash, capillary bleeding, redness, tenderness, scarring, fever, nausea, dizziness, and wheezing. Subjects will be allowed to use analgesics (such as ibuprofen or naproxen) according to the appropriate dose after inoculation and will be provided with analgesics to limit any possible adverse events. Any adverse event will be considered to determine if it is or is not related to the vaccine. All applicable events will be reported to the IRB according to IRB policy 10.2 and to the FDA according to 21 CFR 312.32.

10.1.2 Serious adverse events Serious adverse events are any of the following medical events:
・ Death outcome ・ Immediate threat to life ・ Necessary hospitalization or extension of current hospitalization ・ Congenital anomaly or congenital disorder ・ Or not death outcome, not related to life and death Or other important medical events that do not require hospitalization, based on good medical judgment, these medical events are considered to endanger the subject, and medical or surgical intervention is one of the above outcomes May be considered a serious adverse event if needed to prevent

References for Example 4

All cited publications, patents, and patent literature are hereby incorporated by reference.

Claims (53)

A method of dissolving HPV E6 peptide comprising:
HPV E6 peptide A, which is 20-100 amino acids in length and contains at least 20 consecutive residues of HPV E6 81-115 (residues 81-115 of SEQ ID NO: 1), dissolves the HPV peptide A Prior to the process, at least 50% HPV E6 1-80 (residues 1-80 of SEQ ID NO: 1) and at least 50% HPV E6 116-158, each 10-100 amino acids in length (Residues 116 to 158 of SEQ ID NO: 1) are dissolved in a buffer containing two or more HPV peptides Y in a completely dissolved form, and the peptide A is contained in a completely dissolved form. Creating a final soluble composition comprising in fully dissolved form.   The method of claim 1, wherein the peptide A is acetylated at its amino terminus and amidated at its carboxyl terminus.   The method of claim 1, wherein the HPV peptide A comprises residues 81-115 of SEQ ID NO: 1.   The method of claim 1, wherein said HPV peptide A consists of residues 81-115 of SEQ ID NO: 1.   The method of claim 4, wherein the peptide A is acetylated at its amino terminus and amidated at its carboxyl terminus.   The method according to any one of claims 1 to 5, wherein the buffer has a pH of about pH 3.0 to pH 5.0.   The method according to claim 1, wherein the buffer contains at least 2 mM glutamic acid (salt).   The method of any one of claims 1 to 7, wherein said peptides A and Y collectively comprise all of SEQ ID NO: 1.   The method of claim 1, wherein peptide A consists of residues 81-115 of SEQ ID NO: 1 and said peptide Y is three peptides consisting of residues 1-45, 46-80, and 116-158 of SEQ ID NO: 1.   Each of the peptides A and Y is acetylated at its amino terminus and amidated at its carboxyl terminus, and the pH of the buffer is about pH 3.0 to pH 5.0. 10. The method of claim 9, wherein each is at a concentration of 0.1-20 mg / ml after dissolution.   11. The method of any one of claims 1-10, wherein each of said peptides Y is at least 80% weight / volume concentration of peptide A in said final soluble composition.   The method according to any one of claims 1 to 11, wherein each of peptide A and peptide Y is 0.1 to 5 mg / ml in the final soluble composition. A pharmaceutical composition,
One or more HPV E6 peptides, each of 10 to 100 amino acid residues in length,
-Glutamic acid (salt) at a concentration of 2 to 40 mM,
Trehalose at a concentration of 0.3% to 5% (weight / volume),
Glycine at a concentration of 0.2% to 10% (weight / volume),
Including
-The composition whose pH is 3.0-5.0.   At least one of the one or more HPVs wherein the E6 peptide comprises residues 46-70 of SEQ ID NO: 1, or comprises residues 91-115 of SEQ ID NO: 1, or residues 80-88 of SEQ ID NO: 1 14. The pharmaceutical composition of claim 13 comprising:   14. The pharmaceutical composition of claim 13, wherein the formulation comprises at least 3 HPV E6 peptides, each 10 to 100 amino acid residues in length, collectively comprising at least 50% of the HPV E6 sequence.   16. The pharmaceutical composition according to any one of claims 13 to 15, wherein the composition comprises at least one peptide consisting of residues 1 to 45, 46 to 80, 81 to 115, or 116 to 158 of SEQ ID NO: 1.   The composition comprises a peptide consisting of residues 1-45 of SEQ ID NO: 1, a peptide consisting of residues 46-80 of SEQ ID NO: 1, a peptide consisting of residues 81-115 of SEQ ID NO: 1, and the remainder of SEQ ID NO: 1 16. A pharmaceutical composition according to any one of claims 13 to 15 comprising a peptide consisting of groups 116 to 158.   The pharmaceutical composition according to any one of claims 13 to 17, wherein each of the peptides is acetylated at the amino terminus and amidated at the carboxyl terminus.   The pharmaceutical composition according to any one of claims 13 to 18, further comprising a recall antigen. A pharmaceutical composition comprising:
Comprising HPV E6 peptide A and one or more HPV peptides Y;
HPV E6 peptide A, 20-100 amino acids in length and comprising at least 20 consecutive residues of HPV E6 81-115 (residues 81-115 of SEQ ID NO: 1) dissolves said HPV peptide A Prior to the process, collectively at least 50% of the sequence of HPV E6 1-80 (residues 1-80 of SEQ ID NO: 1) and at least 50% of HPV E6 116-158 (residues 116-158 of SEQ ID NO: 1) In a buffer containing two or more HPV peptides Y each having a length of 10 to 100 amino acids in a completely dissolved form, the peptide A is completely dissolved and the peptide Y is completely A pharmaceutical composition made by a method comprising creating a final soluble composition containing in dissolved form.   21. The pharmaceutical composition of claim 20, further comprising a recall antigen. A method of reducing infection by an individual's human papillomavirus (HPV) or increasing the regression of HPV-related lesions in an HPV-positive individual, comprising:
21. A method comprising administering the pharmaceutical composition of claim 13 or claim 20 to an HPV positive individual in need thereof.   23. The method of claim 22, wherein the pharmaceutical composition further comprises a recall antigen.   23. The method of claim 22, further comprising injecting the recall antigen intradermally.   23. The method of claim 22, comprising injecting the pharmaceutical composition intradermally.   26. The method of any one of claims 22-25, wherein the method increases the regression of HPV-related lesions in an HPV positive individual, wherein the lesion is a malignant tumor.   27. The method of claim 26, wherein the lesion is cervical cancer.   27. The method of claim 26, wherein the lesion is head and neck cancer.   27. A method of increasing regression of HPV-related lesions, wherein the lesion is a tumor of the cervix, vulva, vagina, penis, anus, or pharyngeal cavity. .   26. The method of any one of claims 22-25, wherein the method is to increase regression of HPV-related lesions, wherein the lesion is a highly squamous intraepithelial lesion (HSIL). A method for inducing a T cell response to HPV by reducing infection by an individual's human papillomavirus (HPV) or increasing HPV-related lesion regression in an HPV positive individual, comprising:
Administering to said individual a composition comprising one or more HPV antigens and administering to said individual a recall antigen that is not an HPV antigen, wherein said recall antigen is administered to said one or more HPV antigens of said individual Contacted with
The individual needs a T cell response to the one or more HPV antigens;
The method wherein the one or more HPV antigens are not E6 antigens.   32. does not involve administering a recall antigen that is not an HPV antigen, but produces a more potent T cell response to said HPV antigen in said individual being administered compared to otherwise identical methods. the method of.   33. The method of claim 31 or 32, wherein the composition comprising the one or more HPV antigens also comprises the recall antigen.   32. The step of administering one or more HPV antigens to the individual and the step of administering the recall antigens to the individual comprise injecting the one or more HPV antigens and the recall antigens intradermally. 34. The method according to any one of 33.   35. The method of any one of claims 31 to 34, wherein the one or more HPV antigens comprise an HPV E7 antigen.   The one or more HPV antigens are peptides of 8 to 100 amino acids, 8 to 70 amino acids, 8 to 50 amino acids, or 8 to 40 amino acids in length. One of the methods.   38. The method of claim 36, wherein the peptide is chemically synthesized. A unit dose pharmaceutical composition comprising:
25-110 ug of peptide consisting of SEQ ID NO: 2 25-110 ug of peptide consisting of SEQ ID NO: 3,
25-110 ug of the peptide consisting of SEQ ID NO: 4,
A composition comprising 25 to 110 ug of the peptide consisting of SEQ ID NO: 5 and a recall antigen in a unit dosage form for intradermal injection of 100 to 900 ul.   39. The unit dose pharmaceutical composition of claim 38, wherein the recall antigen is a Candida extract.   40. The unit dose pharmaceutical composition of claim 39, wherein the composition comprises 300 ul CANDIN or equivalent total potency Candida extract.   40. The unit dose pharmaceutical composition of claim 38, wherein the amount is 200-500 ul.   40. The unit dose pharmaceutical composition of claim 38, wherein the composition comprises 30 to 70 ug of each of the peptides.   40. The unit dose pharmaceutical composition of claim 38, wherein said composition comprises about 50 ug of each of said peptides.   44. The unit dose pharmaceutical composition of any of claims 38 to 43, wherein each of the peptides is acetylated at its amino terminus and amidated at its carboxy terminus.   45. A method of treating HPV infection comprising injecting into a skin the unit dose pharmaceutical composition of claim 43 to a patient.   46. The method of claim 45, comprising injecting the unit dose pharmaceutical composition of claim 44 into the patient intradermally at least 3 times consecutively, with 5 to 28 days between each injection.   46. The method of claim 45, comprising injecting the unit dose pharmaceutical composition of claim 44 into the patient intradermally at least 3 times consecutively, with 10 to 21 days between each injection.   48. The method of claim 45, comprising injecting the unit dose pharmaceutical composition of claim 1 into the patient intradermally at least twice consecutively, with 10 to 21 days between each injection.   Injecting said unit dose pharmaceutical composition of claim 1 into said patient intradermally at least 3 times and at most 6 times within a period of 2 years with 5 to 28 days between each injection. 48. The method of claim 46, comprising. A method of treating a disease caused by a microorganism in a mammalian subject comprising:
Administering intradermally to the subject a composition comprising one or more antigens of the microorganism and intradermally administering to the subject a recall antigen that is not an antigen of the microorganism, wherein the recall antigen is administered, Contacting the one or more antigens of the microorganism of interest.   51. The method of claim 50, wherein the microorganism is a virus.   51. The method of claim 50, wherein the microorganism is not a human papillomavirus.   51. The method of claim 50, wherein the recall antigen is a Candida extract.

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